During pregnancy, the mammary epithelium and its supporting vasculature undergo extensive growth and proliferation in preparation for lactation, which is thought to be dependent on vascular endothelial growth factor (VEGF). We investigated the expression of VEGF, using immunohistochemistry and immunoblotting, in the mouse mammary gland during the reproductive cycle. Immunohistochemical studies localized VEGF protein predominantly in the cytoplasm of the mammary epithelium and revealed it to be strongly expressed in late pregnancy and early lactation. In addition, immunoblot analysis revealed a 45-kD VEGF band, corresponding to the homodimer of the VEGF-A₁₆₄ isoform, with increased expression towards the end of pregnancy but no additional increase with the onset of lactation. As the post-lactation period advanced, a dramatic decrease in VEGF was observed in the regressed mammary epithelium. The expression of VEGF suggests that mammary epithelium-derived VEGF may be involved in pregnancy-associated mammary growth and differentiation and angiogenesis, and regulate vascular permeability during lactation in an autocrine or paracrine manner.