Transforming growth factor-beta (TGF-beta) has been implicated in the regulation of ovarian follicle development. Little, however, is known regarding the regulation of TGF-beta expression within the follicle. To investigate this, granulosa and theca cells were isolated from small antral follicles of prepubertal porcine ovaries, maintained in monolayer culture, and treated with gonadotropins or intracellular activators of the protein kinase A and C pathways. TGF-beta secreted into the medium was measured using a proliferation inhibition bioassay with MvLu1 epithelial cells. Over a broad dose range, FSH and LH were ineffective in stimulating TGF-beta secretion relative to controls in granulosa and theca cells, respectively. Additionally, 8-bromo-cAMP, a direct activator of protein kinase A, was ineffective in stimulating TGF-beta secretion in either cell type. In marked contrast, PMA, a stimulator of protein kinase C, dose-dependently stimulated TGF-beta secretion in theca cells. Interestingly, however, PMA had virtually no effect upon granulosa cells. The stimulatory effect of PMA on theca cell TGF-beta secretion was not observed with the inactive derivitive 4 alpha-PMA, and the PMA effect was inhibited by chelerythrine chloride, an inhibitor with high specificity toward protein kinase C. Taken together, these results argue against a direct role of the protein kinase A pathway in the regulation of TGF-beta expression in porcine follicle cells and support direct involvement of the protein kinase C pathway. Moreover, there appears to be marked differences in the regulation of this growth factor between theca and granulosa cells.