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The pattern of steroid synthesis in vitro in ovarian homogenate of immature rats was investigated with [7-³H]pregnenolone as radioactive precursor. The metabolites, progesterone, androstenedione, testosterone and 5-androstane-3,17β-diol were obtained in radiochemically pure form. The steroids were identified by fractionation, partition column and thin-layer chromatography, derivative formation and by crystallization to constant activity. The steroidogenic patterns at the ages of 21 and of 31 days were found to be similar, but different from the pattern reported for adult rat ovaries. At 34 days of age, 1 day before the onset of puberty, the percentage radioactivity converted to progesterone was about ten times higher than that at 31 days. The production in vitro of 5-androstane-3,17β-diol by ovarian homogenates of 34-day-old rats was shown to depend on the concentration of pregnenolone in the incubation mixture. These findings are discussed in relation to the mechanism of the onset of puberty in the female mammal.