To assess the potential role of hormones and growth factors in ovarian follicular growth, we have developed a simple method for the evaluation of DNA synthesis in cultured porcine granulosa cells. Optimal conditions were found to entail newly established cultures that were growth arrested by serum deprivation and then treated with growth-promoting agents. Such treatment resulted in an easily quantitated 3- to 30-fold increase in [3H]thymidine incorporation into trichloroacetic acid-precipitable macromolecules. The radioactivity incorporated was localized to the DNA band on cesium chloride gradients and showed excellent correlation with labeling indices. Insulin, multiplication-stimulating activity, epidermal growth factor (EGF), platelet-derived growth factor, and ovarian follicular fluid were potent stimulators of DNA synthesis in this assay. While EGF and insulin effects were additive, indicating discrete modes of action, the effects of insulin and multiplication-stimulating activity were not additive at maximally effective concentrations. In contrast to the effects of these stimulatory substances, BSA and porcine relaxin were devoid of mitogenic activity under these circumstances. The major trophic hormones implicated in follicular growth, FSH, LH, and estradiol, as well as the cAMP analog 8-bromo-cAMP were without mitogenic effects. Instead, treatment with LH, 8-bromo-cAMP, and the combination of estradiol and FSH resulted in a distinct decrease in DNA synthesis. These data confirm and extend previous evidence of the mitogenic action of insulin-like peptides and EGF, and suggest that the hormones generally believed to regulate granulosa cell replication in vivo lack direct mitogenic effects in vitro.