The role of newly synthesized RNA in the differentiation of granulosa cells isolated from diethylstilbestrol-treated immature rats was studied during culture with actinomycin D. Choleragen-induced LH receptor formation and cGMP production at 48 h were completely inhibited by actinomycin D (greater than or equal to 100 ng/ml) added as late as 20 h after the initiation of culture and were partially reduced by addition of the antibiotic from 30-48 h. In contrast, addition of actinomycin D to freshly prepared cells enhanced choleragen-stimulated cAMP accumulation during the 48-h culture period. This effect was caused by both elevation of adenylate cyclase activity at 3 and 6 h of culture and inhibition of choleragen-induced phosphodiesterase activity during culture. The increase in cAMP production by actinomycin D was confined to the first few hours of culture, since the antibiotic did not enhance cAMP levels when added after 3 h and significantly reduced cAMP accumulation when added from 20-48 h of culture. Actinomycin D inhibited choleragen-stimulated incorporation of [3H]uridine into RNA of freshly prepared cells by 65% and reduced both RNA synthesis and incorporation of [3H]leucine into protein at 20 and 48 h of culture by approximately 90%. In untreated cells, RNA and protein synthesis and phosphodiesterase activity increased to a larger extent from 20-48 h than after choleragen treatment, but did not lead to elevated cAMP levels or LH receptors. These results suggest that the cAMP-induced syntheses of RNA and protein that are specific for increases in cGMP production and LH receptor formation occur predominantly during the second day of granulosa cell culture. In contrast, cAMP production can be markedly altered by changes in RNA and protein syntheses during the first hours of culture.