We have previously shown that TGFB1 inhibits estradiol (E(2)) and progesterone (P(4)) biosynthesis in FSH-stimulated bovine granulosa cells by selective inhibition of steroidogenic enzymes. The objective of this study was to assess the effects of TGFB1 on E(2) and P(4) steroidogenesis in bovine granulosa cells cultured in the absence of FSH and to measure the effects of TGFB1 on cell proliferation and apoptosis in the presence and absence of FSH. Bovine granulosa cells from 2 to 5 mm follicles were cultured in serum-free medium for 2-6 days. In the absence of FSH, the secretion of P(4) increased with time in culture (P<0.05). Addition of TGFB1 for 6 days decreased P(4) secretion and mRNA levels of the P(4) synthesis-associated genes STAR, CYP11A1, HSD3B1, and GSTA (P<0.05). In the absence of FSH, the secretion of E(2) decreased and addition of TGFB1 for 6 days partially reversed this decline and stimulated E(2) biosynthesis, CYP19A1 and HSD17B1 mRNA levels and CYP19A1 activity (P<0.05). Conversely, TGFB1 did not affect HSD17B7 expression and HSD17B-reducing activity. TGFB1 decreased the proportion of cells in the G0/G1 and S+G2/M phases in FSH-stimulated and unstimulated granulosa cells (P<0.05). Furthermore, in the presence or absence of FSH, TGFB1 increased the proportion of cells in apoptosis measured by propidium iodide staining and flow cytometry and confirmed by increased levels of cleaved caspase-3 (P<0.05). Our results therefore indicate that TGFB1 inhibits luteinization in cultured bovine granulosa cells while maintaining an estrogenic phenotype, and this effect was associated with increased apoptosis.