The esterase 6 locus in Drosophila melanogaster is the structural gene for a carboxylesterase (E.C. 3.1.1.1) and is polymorphic for two major electrophoretic variants (slow and fast). Isogenic lines containing X chromosomes extracted from natural populations and substituted into a common genetic background were used to detect unlinked factors that affect the activity of the Est 6 locus. Twofold activity differences of esterase 6 were found among males from these derived lines, which differ only in their X chromosome. These unlinked activity modifiers identify possible regulatory elements. Immunoelectrophoresis was used to estimate quantitatively the levels of specific cross-reacting material in the derived lines. The results show that the variation in activity is due to differences in the amount of EST 6 present. Physiological studies of the control of EST 6 levels reveal that both juvenile hormone and 20 hydroxyecdysone stimulate the production of EST 6 activity in adult males. These results suggest that the effects of X chromosomes on EST 6 activity may be effected by modulating the level of adult hormone titers in Drosophila.