The conversion of alpha-ecdysone to 20-hydroxyecdysone in the midgut of Manduca sexta (L.) was found to be catalyzed by a mitochondrial, cytochrome P450-mediated monooxygenase. The reaction required oxygen and was inhibited by the presence of carbon monoxide. Tricarboxylic acid cycle intermediates, such as succinate, malate, and isocitrate, supported the hydroxylation as did NADPH, NADH, ATP, and ADP. Temperature and pH optima were 30 degrees C and 8.5, respectively. The apparent Km and V values for the ecdysone 20-hydroxylase were 18.3 +/- 6.8 micronM and 46.6 +/- 14.2 pmol per min per mg protein. The midgut mitochondria were found to contain malate dehydrogenase and NAD(P) transhydrogenase. The presence of these enzymes suggests that the tricarboxylic acid cycle intermediates and NADH support the ecdysone hydroxylation indirectly by providing NADPH for the cytochrome P450 system. The content of cytochromes a + a3, b, c + c1, and P450 in midgut mitochondria was determined.