We examine the pathways involved in the lutropin receptor (LHR)-dependent activation of the EGF network using co-cultures of LHR positive granulosa cells and LHR negative test cells expressing an EGF receptor-green fluorescent protein (EGFR-GFP) fusion protein. Activation of the LHR in granulosa cells results in the release of EGF-like growth factors that are detected by measuring the phosphorylation of the EGFR-GFP expressed only in the LHR-negative test cells. Using neutralizing antibodies and real time PCR we identified epiregulin as the main EGF-like growth factor produced upon activation of the LHR expressed in immature rat granulosa cells and we show that exclusive inhibition or activation of the ERK1/2 cascade in granulosa cells prevents or enhances epiregulin release, respectively, with little or no effect on epiregulin expression. These results show that the LHR-stimulated ERK1/2 pathway stimulates epiregulin release.