(a) A selected intermediate point of evolved highly EtOH-tolerant line E9 with an increased mutation rate was evolved during approximately 150 generations on the same percentage of 7% EtOH. After 150 generations the genomic mutation rate, measured in the absence of ethanol, significantly decreased to almost the ancestral mutation rate (mean ± 95% c.i., ***p<0.001, see Materials and methods). (b) Measuring the percentage of dead cells revealed a higher death rate in the START point with high mutation rate compared to the END point with a lower mutation rate (two-sided Student’s t-test, mean ± s.d., n = 3, **p<0.01). (c) To confirm the role of mortality as modulator of cellular mutation rate, the mutS gene was deleted in the END point with a low mutation rate. This deletion caused a significant increase in mutation rate (mean ± 95% c.i., ***p<0.001, see Materials and methods). However, the increase in mutation rate is less pronounced as for the mutS deletion mutant in the clean wild-type background, suggesting the presence of mutations that not only compensate for the original mutator mutation in E9 but also for a deletion of mutS. (d) The number of viable cells decreases significantly for both low and high mutation rate variants during growth on 7% EtOH, although this decrease in the strain with a low mutation rate is less compared to the strain with a high mutation rate (two-sided Student’s t-test, mean ± s.d., n = 3, **p<0.01, ***p<0.001). These results show a lower mortality for a strain with a lower mutation rate, resulting in a competitive advantage in an EtOH environment to which the strain is already adapted.