The c-Src tyrosine kinase has been implicated to play an integral role in modulating growth factor receptor, integrin and steroid receptor function. One class of steroid receptors that c-Src modulates is the estrogen receptor α (ERα). Although there is strong biochemical evidence supporting a role for c-Src in ERα signaling, the consequence of this association is unclear at the biological level. To explore the significance of c-Src in ERα signaling, we studied the development of various reproductive organs that are dependent on ERα in c-Src-deficient mice. We show that the loss of the c-Src tyrosine kinase correlates with defects in ductal development as well as in uterine and ovarian development. Genetic and biochemical analyses of c-Src-deficient mammary epithelial cells also revealed defects in the ability of mammary epithelial cells to activate a number of signaling pathways in response to exogenous estrogen stimulation. Taken together, these studies demonstrate that c-Src plays a role in ERα signaling in vivo.