The object of the present study was to establish conditions (optimal serum concentration and growth factor supplement) for prolonged culture of pig granulosa cells (GC). GC were cultured in the presence of different fetal calf serum (FCS) concentrations (10% and 20%) and one of the following growth factors: leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and stem cell factor (SCF). GC proliferation potential was measured by 3H-thymidine incorporation and telomerase activity whereas cell differentiation by relative transcript abundance of the aromatase gene. In the presence of 10% FCS GC proliferative potential was significantly higher in comparison to control (medium only) after 9 days of culture. Basic fibroblast growth factor significantly increased 3H-thymidine incorporation at all investigated time intervals/points, up to 18 days of culture. Telomerase activity was stimulated by LIF, bFGF and SCF after 3, 6, 9, 12, 15 and 18 days of cultivation while aromatase expression was decreased by all of the applied growth factors, at all investigated time intervals. In conclusion, in the presence of 10% of FCS and bFGF GC can be maintained in culture over prolonged period of time without losing their proliferative potential, telomerase activity and with decreased degree of differentiation measured by aromatase expression.