In order to detect regulatory genetic sites in the autosomes of Drosophila melanogaster, the levels of X-linked glucose-6-phosphate dehydro-genase and autosomally linked α-glycerophosphate and isocitrate dehydrogenases have been monitored in extracts of flies aneuploid for regions of chromosomes II and III. In addition to expected structural gene dosage responses of α-GPDH and IDH, flies hyperploid for several autosome regions were found to display altered levels of one or more of the enzymes studied. While IDH activity was increased in flies hyperploid for segments of both chromosomes II and III, α-GPDH activity was decreased in specific hyperploids for chromosome II regions only. The latter group of segmental aneuploids were normal with respect to levels of chromosome II-linked alcohol dehydrogenase. To test if the observed responses were due to dosage changes of discrete genes lying within the larger effective segments, flies aneuploid for subdivisions of the chromosome segments 21A-25CD, 35A–40, and 70CD–71B were assayed. For two of these large segments so analysed, the apparent effects were attributable to specific small subdivisions, suggesting the presence of discrete regulatory sites within the latter. For the 35A–40 region the α-GPDH effect observed for subdivisions was not sufficient to account for the large α-GPDH decrease seen in flies hyperploid for the large, inclusive region. These observations are discussed with respect to the possible bases of effect of regulatory elements on enzyme activity.