Follicles from hamster ovaries removed at 1000 h of proestrus were incubated for 5 h with the medium changed every hour. Proestrous hamsters were also injected at 1400 h with either 4 mg cycloheximide or saline (control), and follicles were dissected from the ovaries at 1500 h and similarly incubated. During the first hour of incubation, the 1000-h follicles produced picogram amounts of all steroids from progesterone (P4) through estradiol (E2). Thereafter E2, androstenedione (delta) and testosterone (T) accumulation in the medium were reduced by about one-half over the next 4 h, whereas 17 alpha-hydroxyprogesterone (17-OHP) increased to become the dominant steroid. The control follicles incubated at 1500 h produced steroids in nanogram amounts and the granulosa cells in vitro now produced C21 steroids, especially P4. In contrast, throughout the 5-h incubation, the follicles of cycloheximide-treated hamsters accumulated considerably reduced amounts of C21 steroids and this was reflected in granulosal production of P4 at one-half to one-tenth of control levels. The profiles for accumulation of C19 and C18 steroids by the intact follicles of both groups were superimposable over the 5-h period. Thecal production of delta was unaffected by injection of cycloheximide at 1400 h, which suggests that the theca requires the continued presence of high concentrations of cycloheximide to affect steroidogenesis. Incubation of granulosa cells from cycloheximide-exposed follicles with 10 ng of various steroid precursors restored P4, 17-OHP, E2 and estrone (E1) to the same levels as controls, indicating that the steroidogenic enzymes from 3 beta-ol-dehydrogenase through aromatase were unaltered and therefore the lack of sterol precursors was the critical step affected by cycloheximide. Incubation of cycloheximide-treated granulosa cells with 25-hydroxycholesterol led to a 3-fold increase in P4 levels which are still only one-half of control values. This points to decreased accessibility of cholesterol to mitochondrial side-chain cleavage as one of the key events blocked by cycloheximide. The experiments show that after exposure to the proestrous surge of gonadotropins, there is normally a rapid recruitment of the granulosal compartment as a source of C21 steroids and this is a protein-dependent process.