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Five enzyme activities in midgut cytosol of Manduca sexta last instar larvae are potentially involved in the interconversion of 3β-hydroxyecdysteroids, 3-oxoecdysteroids, and 3α-hydroxyecdysteroids. A Sephadex G-25-filtered high-speed supernatant was used to determine some of the characteristics of the corresponding enzymes. The pH optima of ecdysone oxidase and NADH-dependent 3-oxoecdysteroid 3α-reductase were 7.5, the pH of the midgut cytosol was 7.9. The apparent kinetic parameters for the NADH-dependent 3α-reductase were Km (for NADH) = 80.8 ± 10.8 μM and Vmax = 0.58 ± 0.30 nmol/min/mg protein, and for the NADPH-dependent 3-oxoecdysteroid 3β-reductase, Km (for NADPH) = 19.3 ± 2.5 μM and Vmax = 4.39 ± 0.40 nmol/min/mg protein. NAD+ and NADP+ inhibited the enzymatic 3-oxoecdysteroid reductions, but the reactions were not reversible (i.e. no conversion of ecdysone or 3-epiecdysone to 3-dehydroecdysone). Sodium chloride (0.2 M) inhibited the 3α-reductase activity with NADH and strongly increased the 3α-reductase activity with NADPH.