Recently, we found that the activity of malate dehydrogenase (MDH), one of the key enzymes in the biosynthesis of lipids, is regulated in Drosophila melanogaster by juvenile hormone (JH) and JH analogues. Here we report that the response of MDH to JH, however, depends on the developmental stage of the animal in relation to its endogenous titre of ecdysteroids. During the interecdysial period of the last instar, when endogenous ecdysteroids are low, MDH rapidly responded to JH by increasing activity, while little or no response was monitored in wandering (post-feeding) larvae, i.e., after a pulse of ecdysteroids. Activity of MDH in ecd 1 and su(f) ts67g , two ecdysteroid-deficient mutants of Drosophila, increased during a temperature shift to 29°C as compared to wild-type controls, and was sensitive to administration of JH. In contrast, when ecd 1 or su(f) ts67g larvae during restrictive conditions were fed on a 20-hydroxyecdysone diet, JH was unable to increase the activity of the MDH enzyme. However, when JH was applied prior to 20-hydroxyecdysone feeding, the activity of MDH was significantly stimulated. Two phases of MDH response to JH, dependent on ecdysteroids, could be observed. The first phase was short and independent of RNA and protein syntheses. But activation of MDH in later stages of the response was inhibited by actinomycin D and cycloheximide, indicating that MDH might be regulated by JH at the transcriptional and/or translational level. Arch.