The biosynthesis of testosterone within the testis has been shown to take place along different metabolic pathways in different species. In the testis from elderly men with prostatic carcinoma the delta 5-metabolic pathway has been shown to be preferred. Less is known about the preferred pathway in younger adults. In this study, testicular biopsy specimens from 33 adults (ranging from 27 to 35 years of age) were incubated with both tritiated pregnenolone and progesterone. The production of testosterone from these precursors was determined in vitro and was found to be significantly higher from pregnenolone than from progesterone. This indicates that pregnenolone was initially mainly converted along the delta 5-metabolic pathway to 17 alpha-hydroxypregnenolone and probably further to dehydroepiandrosterone before it was subsequently transformed to a delta 4-metabolite. Similar results were obtained in incubation studies performed with testicular tissue from eleven elderly men (ranging from 60 to 83 years of age). In conclusion, studies of the testosterone production in vitro suggest that the delta 5-metabolic pathway is preferred in the testis from younger as well as elderly men.