We evaluated the effects of transforming growth factor β1 (TGFβ1), alone or in combination with FSH and estradiol, on DNA synthesis in primary cultures of immature rat granulosa cells. ³H-Thymidine incorporation was significantly stimulated by TGFβ1 (5.6-fold). This effect was enhanced by FSH (20 ng/ml, 27.7-fold) or estradiol (100 ng/ml, 13.4-fold) or by a combination of both hormones (59.2-fold). Measurement of TGFβ bioactivity showed the presence of significant amounts of TGFβ in conditioned medium from granulosa cell cultures, and most of the activity was present in the latent form. FSH alone or in combination with estradiol produced a marked suppression of the production of latent and active TGFβ. Activated conditioned medium from control cultures of granulosa cell elicited a 1.4-fold increase in thymidine incorporation. This effect was markedly amplified by FSH (3-fold) and estradiol (4.3-fold) and by a combination of both (8.7-fold). The peptide containing the cell-binding domain of fibronectin (RGDSPC) partially inhibited thymidine incorporation stimulated by TGFβ1. Fibronectin did not synergize with FSH, and the interaction between TGFβ1 and FSH was even observed in the presence of this protein. The conclusions reached were as follows: 1) TGFβ1 is an autocrine stimulator of rat granulosa cell DNA synthesis, 2) FSH and estradiol produce a suppression of latent and active TGFβ production but markedly amplify TGFβ action, presumably at a postreceptor level, and 3) the stimulatory effects of TGFβ1 may be only partly mediated by the increased fibronectin secretion.