The effects of nicotine and cotinine, the major metabolite of nicotine, on testosterone production in rat testis were investigated. Rat Leydig cells were isolated and incubated with nicotine and cotinine. Nicotine produced a dose dependent increase in progesterone levels and a dose-dependent decrease in androstenedione and testosterone concentrations. Cotinine produced a dose-dependent increase in progesterone and androstenedione and a dose-dependent decrease in testosterone levels. The effects of nicotine and cotinine on rat testis mitochondrial cholesterol side chain cleavage enzyme and microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase, 17 alpha-hydroxylase, 17,20-lyase and 17-ketosteroid reductase were examined. Nicotine competitively inhibited 17 alpha-hydroxylase (apparent Ki = 30 microM) and 17,20-lyase (apparent Ki = 18 microM). Cotinine competitively inhibited 17-ketosteroid reductase (apparent Ki = 46 microM). The addition of nicotine to preparations of rat testis microsomes yielded a Type II cytochrome P-450 binding spectrum. We conclude that nicotine and cotinine competitively inhibit multiple steps in testosterone biosynthesis.