Enhanced accumulation of follicular PGF2 alpha with respect to PGE2 during the later phase of the preovulatory period is an apparent prerequisite for ovulation in sheep. Prostaglandin (PG) E2-9-ketoreductase is the enzyme that converts PGE2 into PGF2 alpha. Expression of activity of this enzyme by tissue homogenates of preovulatory ovine follicles was assessed. Homogenates were incubated in the presence of tritiated PGE2. Prostaglandin F2 alpha (i.e., product) was separated from PGE2 by Sephadex chromatography and quantitated by liquid scintillation counting. Progesterone in follicular fluid was measured by RIA. Follicular activity of PGE2-9-ketoreductase and content of progesterone increased approximately sixfold as the time of ovulation approached. Formation of PGF2 alpha from PGE2 was not influenced by inhibition of follicular synthesis of prostaglandins by indomethacin, nor did such treatment affect follicular production of progesterone. Inhibition of follicular synthesis of progesterone by isoxazol suppressed enzymatic conversion of PGE2 into PGF2 alpha; this effect was reversed by progesterone. It appears that progesterone plays an intrafollicular role in induction of activity of PGE2-9-ketoreductase in sheep.