The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.