Seven enzyme activities were measured in Drosophila melanogaster lines in which spontaneous mutations had accumulated over about 300 generations under the minimum pressure of natural selection. These enzymes included alcohol dehydrogenase (ADH), alpha-glycerol-3-phosphate dehydrogenase (alpha GPDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD) and alpha-amylase (AMY). A significant genetic variance was observed for some enzyme activities. The mutations which alter the enzyme activities are called modifier mutations. The magnitudes of the genetic variance in modifier mutations differed greatly among enzymes but were often similar between two series of mutation accumulation lines (AW and JH). This may therefore indicate that the number of modifiers is specific for each enzyme system. The modifier mutation rate is suggested to be one of the clues for assessing the maintenance mechanism of protein polymorphism in natural populations.