The first step in the biosynthesis of ecdysteroids by Manduca sexta prothoracic glands, the conversion of cholesterol to 7-dehydrocholesterol, is mediated by an enzyme with characteristics of a microsomal cytochrome P-450, i.e. sensitivity to CO and fenarimol, and a requirement for NADPH. The enzyme responsible for hydroxylation at C-25 of the putative 3-dehydroecdysone precursor, 14-hydroxy-5 beta-cholest-7-en-3,6-dione, is also microsomal, while those mediating hydroxylations at C-22 and C-2 of 3,14,25-trihydroxy-5 beta-cholest-7-en-6-one are mitochondrial. Indirect evidence revealed that the steps between 7-dehydrocholesterol and the trideoxyecdysteroids occur in the mitochondria, suggesting that extensive shuttling of intermediates between the endoplasmic reticulum and mitochondria takes place in the prothoracic gland cell during ecdysteroid biosynthesis. During the fifth larval instar, cholesterol 7,8-dehydrogenase activity is evident from days 2 to 9, while the conversion to [3H]ecdysteroids is not significant prior to the ecdysteroid commitment peak on day 4. Terminal hydroxylase activity shows little change throughout the instar. These data support the hypothesis that regulation of the biosynthetic pathway by PTTH occurs at the step immediately following the formation of 7-dehydrocholesterol. The steroid biosynthesis inhibitor, fenarimol, has been shown to inhibit each of these P-450 enzymes, as well as fat body ecdysone 20-monooxygenase, with an I50 of 10(-4) M in disrupted glands, suggesting that it is a general P-450 inhibitor. The secretion of ecdysteroids by the glands in vitro is very sensitive to fenarimol, i.e. I50 of 10(-6) M. RH5849, 1,2-dibenzoyl-1-tert-butylhydrazine, fails to inhibit any of these prothoracic gland reactions, yet strongly inhibits fat body ecdysone 20-monooxygenase activity. This suggests that RH5849 is a specific ecdysteroid substrate/product mimic in this reaction.