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Prostaglandin F 2α (PGF 2α ) did not alter the in vitro biosynthesis of progesterone by slices of luteinized rat ovaries when used in concentrations from 1 to 10,000 ng/ml of incubation medium; likewise, PGF 2α did not affect the incorporation of acetate-1- 14 C into progestins. PGF 1α , 15-keto PGF 2α , and PGE 1 did not alter the biosynthesis of progesterone by luteinized rat ovaries; PGE 2 inhibited the production of progesterone when used at a concentration of 10 μg/ml, but not at lower doses. PGF 2α in combination with luteinizing hormone (LH) enhanced the metabolism of progesterone to 20α-hydroxypregn-4-en-3-one in luteinized rat ovaries. Interestingly, PGF 2α , at a high concentration of 10 μg/ml, did stimulate progesterone biosynthesis by slices of ovarian tissue from immature rats hormonally primed to simulate “pseudopregnancy,” suggesting a steroidogenic action of prostaglandins on the ovarian follicular or interstitial cell. PGF 2α (10 μg/ml) did not stimulate the in vitro biosynthesis of progesterone or 20α-hydroxypregn-4-en-3-one by slices of rabbit corpora lutea or rabbit ovarian interstitial tissue. It is concluded that prostaglandins do not stimulate progestin biosynthesis in rat luteal tissue.