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The ovarian tissue components of the pregnant chinchilla were incubated with equimolar amounts of [7-³H]pregnenolone and [4-¹⁴C]progesterone. The greater contribution by [7-³H]pregnenolone than by [4-¹⁴C]progesterone towards the formation of 17-hydroxyprogesterone and androstenedione, and the relatively high yields of 17-hydroxypregnenolone and dehydroepiandrosterone showed that both the 4-ene and 5-ene pathways of steroid metabolism were used in the interstitial tissue. No significant amount of 17-hydroxylation was observed in the primary and accessory corpora lutea. The results of kinetic investigations using [7-³H]pregnenolone as substrate also demonstrated a precursor—product relationship between dehydroepiandrosterone and androstenedione in the interstitial tissue, but this was not apparent in the luteal tissue. The results indicated that the interstitial tissue was capable of synthesizing progesterone and oestrogens as major products, and that the lack of 17-hydroxylation in the luteal tissue was a controlling factor ensuring the synthesis of progesterone as its principal hormonal product. A small amount of [4-¹⁴C]dehydroepiandrosterone was always isolated with a much larger amount of the tritiated compound. This implied the conversion of ¹⁴C-labelled 4-en-3-oxosteroids into 5-ene-3β-hydroxysteroids which has generally been regarded as impossible. The isolation of this product, which may be an artifact, and the possibility that progesterone and oestrogens may be synthesized by different cells (granulosa and theca lutein cells) in the corpus luteum, or that there may be a third pathway for oestrogen synthesis, as suggested by the results of the kinetic experiments, are discussed.