Eight ewes each with an autotransplanted ovary received infusions of tritium-labelled pregnenolone (41 muCi/hr) for 8 hr into the artery supplying the ovary, together with prostaglandin (PG) F-2alpha (30 mug/hr) for 3 hr beginning 2 hr after the start of the pregnenolone infusion. All animals exhibited oestrus 2-3 days after the start of the experiment. During the PGF-2alpha infusion blood flow through the ovaries was increased by 13%, but subsequently returned to pre-infusion levels. Secretion rates of endogenous progesterone and 20alpha-hydroxypregn-4-en-3-one dropped rapidly 5 hr after the PGF-2alpha infusion had started from values of 250 mug/hr and 25 mug/hr to values below 60 mug/hr and 8 mug/hr, respectively. At this time the conversion of radioactive pregnenolone to progesterone was reduced by 50% of its initial value, but the secretion of endogenous pregnenolone and the formation of radioactive metabolites other than progesterone were not diminished. In 4 control animals, which received pregnenolone only, no changes in ovarian blood flow, steroid secretion rates, or in the conversion of labelled pregnenolone were observed. These results suggest a possible involvement of PGF-2alpha in the regulation of progesterone biosynthesis by an action on the 3beta-hydroxysteroid oxidoreductase-delta5(-4) isomerase enzyme system.