This study was designed to investigate whether prolactin (PRL)affects coordinated regulation of tissue plasminogen activator (tPA) and plasminogen activator inhibitor type-Ⅰ (PAI-Ⅰ) gene expression in rat granulosa cells in vitro. Several methods, such as SDS-PAGE, immunoblot etc. were used to detect the effect of PRL on tPA and PAI-Ⅰ gene expression. The results demonstrated: (1) PRL increases PAI-Ⅰ mRNA production in cultured granulosa cells. Inclusion of follicle stimulating hormone (FSH) with PRL has a synergistic effect on the increase of PAI-Ⅰ mRNA levels. After 48 h culture in the presence of FSH with PRL, a 7.8-fold increase in PAI-Ⅰ mRNA levels is observed as compared with PRL alone. The synergistic increase in PAI-Ⅰ mRNA levels occurs in a dose- and time-dependent manner; (2) the increase in PAI-ⅠmRNA synthesis in the cells by PRL alone, or PRL in combination with FSH, is well correlated with the changes of PAI-Ⅰ activity levels in the conditioned media; (3) PRL in culture also decreases FSH-induced tPA activity level in a dose-dependent fashion. The decrease in FSH-induced tPA activity level by PRL is correlated with an increase in the amount of PA-PAI-Ⅰ complexes in the conditioned media. This suggests that the decline of tPA activity is related to neutralization of tPA by the increased PAI-Ⅰ activity in the media.