Administration of an ovulatory dose of LH (10 micrograms, i.v.) to PMSG (4 i.u., s.c.)-primed immature rats increased ovarian pregnenolone levels 5-fold and ovarian progesterone levels 40-fold within 6 h and the levels, although fluctuating, remained elevated for 72 h. Serum progesterone levels mimicked those of the luteal phase in the normal cycle. The sustained increase in steroidogenesis was accompanied by a decrease in both basal and cAMP-stimulatable ovarian cholesterol ester hydrolase activity and a net increase in ovarian cholesterol ester content. Ovarian free cholesterol levels were essentially unchanged during the 72 h study. LH does not, therefore, chronically stimulate steroidogenesis by providing additional substrate for the steroidogenic enzymes through activating cholesterol ester hydrolase to bring about hydrolysis of cholesterol esters. Moreover, ovarian cholesterol esters are unlikely to be the primary source of cholesterol utilized to support luteal steroidogenesis. Studies with isolated mitochondria suggested that the mechanisms by which LH stimulated steroidogenesis were by (a) stimulating mitochondrial pregnenolone production probably by facilitating the intramitochondrial movement of cholesterol of the site of side-chain cleavage, and (b) promoting the metabolism of pregnenolone to progesterone.