In order to establish an immortalized granulosa cell line and to investigate the potential mechanisms of immortalized cell proliferation, simian virus (SV) 40 was used to infect porcine granulosa cells from small follicles (1-2 mm in diameter), and one colony was selected after four weeks of culture. The colony was digested with trypsin and the cells were cultured for more than 300 days (named PGV). The SV40 large T antigen gene and its products were confirmed in immortalized cells by Southern blotting and immunohistochemistry. Progesterone production was not detected in the conditioned culture media with follicle-stimulating hormone (FSH) and forskolin, possibly due to the lack of P450scc gene transcription as examined by Northern blotting. PGV cells responded significantly to the stimulation of sera (fetal bovine and horse sera) and protein kinase C (PKC) stimulators (PMA and OAG), while PKC inhibitors (staurosporine and calphostin C) blocked both sera and PKC stimulation. Phospholipase C (PLC) and phosphatidic acid phosphatase (PAP) inhibitors (U73122 and propranolol) significantly reduced PGV cell proliferation, while PMA restored PLC and PAP inhibition. These data suggest that diacylglycerol (DAG) is produced in PGV cells by PLD as well as by PLC, and that DAG then activates PKC stimulating the PGV cell cycle through yet unknown mechanisms. Thus, an immortalized granulosa cell line is very useful to study granulosa cells in vitro, as the cells are homogeneous and are a functionally defined population.