Injecting 2 or 4 mg of cycloheximide (cyclo) at the onset of the proestrous release of gonadotropins prolongs the estrogen (E2) surge, diminishes progesterone (P4) secretion, and prevents ovulation by 0900 h of the next morning (Saidapur and Greenwald, 1981). The present study was designed to determine the effects of 0, 2, 4, or 8 mg cyclo injected at 1400 h proestrus (Day 4) on ovarian protein synthesis and other parameters. Ovulation was delayed until 1400 h estrus by 2 mg cyclo or prevented by 8 mg, and the latter treatment resulted in the death of all animals by 48 h. After 4 mg cyclo, ovulation was delayed in some animals, but the most characteristic feature was the development of large cystic follicles that ultimately transformed into corpora hemorrhagica. All animals lived after the injection of 4 mg cyclo. Ovaries collected 2, 8, 16, or 24 h after treatment were incubated with [3H]leucine for 1 h to assess the effects of cyclo on protein synthesis. Injection of phenobarbital at 1300 h proestrus, which blocks follicle-stimulating hormone (FSH) and luteinizing hormone (LH) surges, reduced ovarian protein synthesis at 1600 h to 61% of the control value. The incorporation of [3H]leucine was reduced to 75%, 37%, and 35% of the 1600-h control value by 2, 4, and 8 mg cyclo, respectively, but without affecting surge levels of FSH and LH. However, by 0600 h estrus, protein synthesis was increased significantly in all the cyclo-treated groups, which provides insight into the half-life of the compound (approximately equal to 8 h for 2-4 mg cyclo). At 1600 and 2200 h proestrus cyclo resulted in serum FSH and LH levels similar to controls, but increased serum prolactin and prolonged E2 levels at Day 4 of 2200 h and decreased serum P4 at both times. The second surge in FSH, which is in progress by 0600 h estrus, was abolished by 4 or 8 mg cyclo but not by the 2-mg dose. This is the first time for any species that ovarian protein synthesis has been measured in the proestrous normal or cyclo-treated animal. We conclude for the hamster that 4 mg cyclo is the optimal dose for blocking ovarian protein synthesis and ovulation and inducing formation of cystic follicles.