Genistein affects reproductive processes in animals. However, the mechanism of its action is not fully elucidated and differs among species. The objectives of the current study were: 1/ to establish an in vitro model of granulosa cell culture for studying the intracellular mechanism of phytoestrogen action in porcine ovary; 2/ to determine an in vitro effect of genistein on basal and FSH-stimulated P(4) and E(2) production by porcine granulosa cell populations (antral, mural, total) isolated from large, preovulatory follicles. Granulosa cells were isolated from large (> or =8 mm), preovulatory follicles and separated into antral and mural cell subpopulations. Cells were allowed to attach for 72 h (37 degrees Celsius, 10% serum, 95% air/5% CO2) and than cultured for next 48 hours with or without serum (0, 5 and 10%), FSH (0, 10 or 100 ng/ml) and genistein (0, 0.5, 5 or 50 microM). Basal P(4) and E(2) production did not differ among antral, mural and unseparated granulosa cells isolated form porcine preovulatory follicles. Only mural cells tended to secrete less P(4) and E(2) than other cell populations. FSH stimulated P(4) production in a dose dependent manner in all cell populations and culture systems. Genistein inhibited in a dose dependent manner basal and FSH-stimulated P(4) production by antral, mural and unseparated granulosa cells. However, genistein did not affect E(2) production by granulosa cells. In addition, viability of porcine granulosa cells was not affected by the pyhytoestrogen except the highest dose of genistein. It appears that genistein may be involved in the regulation of follicular function in pigs. Moreover, unseparated porcine granulosa cells may provide a suitable in vitro model for studying the intracellular mechanism of phytoestrogen action in porcine ovary.