Female rats of the Sprague-Dawley strain were used. Two pituitaries from 9-week old rats were grafted in both kidneys of 21-day old rat to induce hyperprolactinemia. All rats with or without pituitary isografts were hypophysectomized on day 26. Starting from day 29, the rats in groups of 8-11 were injected daily with 5 micrograms NIH-LH-S19 or saline for 3 days. Ovarian homogenates from these rats on day 32 were incubated with [14C]4-androstene-3,17-dione or [3H]progesterone and steroid metabolism was estimated. In the hypophysectomized rat ovary, the 5 alpha-reductase activity was stimulated significantly by LH. Although pituitary isograft alone had no stimulative effect on 5 alpha-reductase activity of the hypophysectomized rat ovary, concomitant treatment with LH and pituitary isograft (prolactin) had an additive effect. Formation of the sum of C19-steroids from progesterone in the hypophysectomized rat ovary was stimulated markedly by LH but reduced slightly by prolactin. The LH-induced production of C19-steroids from progesterone was inhibited markedly by prolactin. These results indicate that prolactin treatment inhibits basal and LH-induced production of C19-steroids from progesterone but stimulates LH-induced 4-ene-5 alpha-reductase activity in immature rat ovary.