The impact of lowering the ovarian L(iver)-type lipase activity on cholesterol homeostasis in the ovaries was studied in superovulated rats. L-type lipase activity increased rapidly after injection with chorionic gonadotrophin (HCG) (day 0), its activity remained high between days 3 and 8. During this period plasma progesterone and 20 alpha-hydroxyprogesterone were raised. The ovarian content of unesterified cholesterol remained constant during this period while cholesterol esters increased. Lowering of the L-type lipase activity by in vivo treatment with anti-liver lipase (ALLA) during 4-5 h did not affect plasma hormones or ovarian cholesterol contents. However, de novo cholesterol synthesis in the ovaries was significantly increased by about 40%. After pretreatment of the rats with aminogluthetimide, ALLA administration led to a 250% increase in de novo cholesterol synthesis in the unesterified cholesterol fraction, but was without effect on plasma hormones and on the ovarian cholesterol content. Administration of the cholesterol synthesis inhibitor Simvastatin led to a 25% lowering in ovarian cholesterol synthesis without effect on plasma hormones or ovarian cholesterol content. Additional administration of ALLA affected only the plasma progesterone (-30%). These results indicate that L-type lipase is involved in ovarian cholesterol homeostasis.