(A) An aversive taste memory assay was used to assess fatty acid taste discrimination in female w1118 flies at a 1% concentration. First, initial responses to a short-, medium-, or long-chain fatty acid were assessed (Pretest). Next, flies were trained by pairing this fatty acid with quinine presentation immediately following tastant application (Training). Proboscis extension response (PER) in response to either the same or different fatty acid was then tested in the absence of quinine (Test). In control experiments (Naïve), the same procedure was followed, but quinine was not applied to the proboscis. (B) The pairing of medium-chain hexanoic acid (6C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER response to 6C was significantly lower in trained flies compared to naïve flies (p<0.0001), but there was no difference in PER to short-chain valeric acid (5C; p=0.6864). Restricted maximum likelihood (REML): F1,80 = 7.329, p=0.0003, with Sidak’s test for multiple comparisons; N = 40–42. (C) The pairing of medium-chain hexanoic acid (6C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER response to 6C was significantly lower in trained flies compared to naïve flies (p<0.0001), but there was no difference in PER to long-chain nonanoic acid (9C; p=0.3346). REML: F1,64 = 6.296, p=0.0146, with Sidak’s test for multiple comparisons; N = 33. (D) The pairing of short-chain valeric acid (5C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER response to 5C was significantly lower in trained flies compared to naïve flies (p=0.0014), but there was no difference in PER to long-chain nonanoic acid (9C; p=0.0789). REML: F1,46 = 2.721, p=0.0105, with Sidak’s test for multiple comparisons; N = 24. (E) The pairing of short-chain butanoic acid (4C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER to both 4C and short-chain valeric acid (5C) was significantly lower in trained flies compared to naïve flies (4C: p<0.0001; 5C: p<0.0001). REML: F1,38 = 33.67, p<0.0001, with Sidak’s test for multiple comparisons; N = 20. (F) The pairing of medium-chain hexanoic acid (6C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER to both 6C and medium-chain heptanoic acid (7C) was significantly lower in trained flies compared to naïve flies (6C: p<0.0001; 7C: p<0.0001). REML: F1,81 = 45.88, p<0.0001, with Sidak’s test for multiple comparisons; N = 41–42. (G) The pairing of medium-chain hexanoic acid (6C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER to both 6C and medium-chain octanoic acid (8C) was significantly lower in trained flies compared to naïve flies (6C: p<0.0001; 8C: p<0.0001). REML: F1,65 = 32.76, p<0.0001, with Sidak’s test for multiple comparisons; N = 33–34. (H) The pairing of medium-chain heptanoic acid (7C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER to both 7C and medium-chain octanoic acid (8C) was significantly lower in trained flies compared to naïve flies (7C: p<0.0001; 8C: p<0.0001). REML: F1,72 = 33.67, p<0.0001, with Sidak’s test for multiple comparisons; N = 37. (I) The pairing of long-chain decanoic acid (10C) and quinine (red) results in a significant reduction in PER compared to naïve flies. After training, PER to both 10C and long-chain nonanoic acid (9C) was significantly lower in trained flies compared to naïve flies (10C: p<0.0001; 9C: p=0.0015). REML: F1,38 = 33.23, p<0.0001, with Sidak’s test for multiple comparisons; N = 20. Error bars indicate ± SEM. **p<0.01; ***p<0.001; ****p<0.0001.