Two pituitaries from 7-week-old female rats were grafted under the capsule of the left kidney of 50-day-old male rat to induce hyperprolactinemia. All of the pituitary-grafted and sham-operated rats were hypophysectomized at 56 days of age. The hypophysectomized rats in groups of 4 were given daily sc injections of saline or 9 micrograms NIADDK-ovine-(o)LH-23 for 4 and 5 days starting from days 58 and 70, respectively (short and long term hypophysectomized groups). The metabolism of [3H]progesterone or [14C]androstenedione by testicular homogenates, concentrations of testosterone and 5 alpha-androgens (androsterone plus 5 alpha-androstane-3 alpha, 17 beta-diol) in the serum and testes, and testicular LH receptors were estimated. Hypophysectomy caused significant decreases in testicular enzyme activities per gram of tissue, androgen production, and testicular LH receptors. In the testes of hypophysectomized rats, LH treatment significantly stimulated 5 alpha-reductase and 17-hydroxylase activities. Although pituitary grafts alone showed little or no effect on these testicular enzyme activities, hyperprolactinemia induced by the grafts markedly enhanced the LH-stimulated 5 alpha-reductase activity in both groups, especially in the long term hypophysectomized group. Therefore, androsterone and 5 alpha-androstane-3 alpha,17 beta-diol were shown to be the major C19-steroid products (immature type of testicular androgen production) in the LH- and PRL-stimulated testes of long term hypophysectomized adult rats. On the other hand, hyperprolactinemia was associated with a significant inhibition and a slight increase of the LH-stimulated 17-hydroxylase activities in the short and long term hypophysectomized groups, respectively. This difference can be attributed to both a PRL-induced increase in testicular LH receptors and a PRL-induced inhibition of 17-hydroxylase via a postreceptor mechanism(s). The present findings demonstrate for the first time that PRL directly stimulates LH-induced 5 alpha-reductase activity in the testes. It appears that PRL may play a role in the increased production of 5 alpha-C19-steroids and the parallel decrease of testosterone production in immature rat testes.