A surge in luteinizing hormone (LH) triggers physiological changes within the ovarian follicles, including reprogramming to induce terminal differentiation of the granulosa cells (GCs). Cytokines are members of a large regulatory network that resides in the ovaries and are involved in the regulation of steroidogenesis and gamete production. Recently we found that interferon-alpha (IFN-alpha) was overexpressed in LH-treated preovulatory GCs, as determined by a microarray analysis. In this study, we evaluated the expression of IFN-alpha and its role in the differentiation of rat preovulatory GCs. Rat GCs were treated with LH in vitro or human chorionic gonadotropin (hCG) in vivo, both of which are well-known inducers of differentiation, and IFN-alpha production and cell differentiation were determined. Stimulation of rat primary GCs with LH or hCG increased expression of IFN-alpha. LH treatment led to increased phosphorylation of PI3-K and extracellular signal-regulated kinase (ERK), and specific inhibitors for PI3-K and ERK suppressed the LH-induced IFN-alpha expression in preovulatory GCs. Furthermore, treatment with anti-rat IFN-alpha blocking antibody delayed the LH-induced differentiation of GCs and suppressed the expression of ovulation-related genes, including progesterone receptor (PR) and steroidogenic acute regulatory protein (StAR). These results indicate that LH induces IFN-alpha expression in preovulatory GCs via a PI3-K/ERK signaling pathway and that interferon-alpha production may be involved in the LH-induced differentiation of preovulatory GCs in rats.