ABSTRACT Decapsulated testes of rats, guinea pigs and rabbits were incubated with or without labelled precursors and the steroids formed under various experimental conditions were analysed. Also a decapsulated human testis was incubated with labelled acetate. Cholesterol, testosterone and androstenedione were isolated in a radiochemically homogeneous form following the incubation of carbon-14-labelled sodium acetate with decapsulated testes of all 3 animal species. No other labelled steroid was detected following the incubation of rat testes. Guinea pig testes also converted labelled acetate to pregnenolone. Rabbit and human testes converted labelled acetate to cholesterol, pregnenolone, dehydroepiandrosterone, androstenediol, testosterone and androstenedione. When decapsulated testes of rats, guinea pigs and rabbits were incubated with carbon-14 labelled pregnenolone as a precursor, radiochemically homogeneous progesterone, 17-hydroxypregnenolone, dehydroepiandroster-one, androstenediol, androstenedione and testosterone were isolated in each experiment. Using radioimmunoassay techniques, preformed steroids together with steroids formed from endogenous precursors were analysed following the incubation of rat, guinea pig and rabbit testes in the absence and in the presence of human chorionic gonadotrophin (HCG). Marked (seasonal?) variations were observed between the results of experiments conducted at different times. Incubation of guinea pig testes in the absence as well as in the presence of HCG resulted in a major increase in pregnenolone levels. No such finding was encountered when rat and rabbit testes were incubated. The addition of HCG resulted in at least a 10-fold increase in testosterone formation by the testes of all three species. The addition of HCG to the incubation medium induced a marked rise in dihydrotestosterone levels in the rabbit testes but had no effect whatsoever on the levels of this steroid in guinea pig testes. It is concluded that considerable species differences exist in the steroid metabolism of decapsulated testes incubated in vitro.