The present study examined changes in steroidogenic enzyme activities which metabolize testosterone or dihydrotestosterone between days 21–73 of maturation in Band 2 and Band 3 cells isolated by centrifugation of rat testicular interstitial cells on metrizamide density gradients. 5α-reductase and 17β-hydroxysteroid dehydrogenase activities increased progressively in Band 2 and Band 3 cells between days 21–35 of maturation, then both enzyme activities declined to reach low levels in adult Band 2 and Band 3 cells. The significantly higher activities of both enzymes in Band 3, which contains a greater concentration of Leydig cells at each age, suggest their localization in Leydig cells. 5α-androstane-3α- and 3β-hydroxysteroid dehydrogenase activities increased in both Band 2 and Band 3 cells between days 21–50 of maturation and remained elevated; however, dihydrotestosterone was metabolized primarily to 5α-androstane-3α,17β-diol in Band 2 cells, while 5α-androstane-3β,17β-diol was the major metabolite of dihydrotestosterone in Band 3 cells. These studies suggest that testosterone accumulation during sexual maturation can be influenced by changing patterns of 5α-reductase and 17β-hydroxysteroid dehydrogenase activities which metabolize testosterone, and of 5α-androstane-3α- and 3β-hydroxysteroid dehydrogenase activities which metabolize dihydrotestosterone in both Band 2 and Band 3 cells.