The hormonal stimulation of mammary gland morphogenesis is believed to occur through growth factor receptor signaling pathways. To determine the importance of the epidermal growth factor receptor (EGFR) pathway, we examined extracts of inguinal mammary glands from prepubertal and pubertal mice for tyrosine-phosphorylated EGFR and other erbB receptors. Tyrosine phosphorylation of both EGFR and erbB-2 was detected in normal female BALB/c mice at 5-6 weeks of age, but not during the prepubertal stage, e.g., 24 days of age. Treatment of mice with estradiol or epidermal growth factor also stimulated the formation of mammary EGFR/erbB-2 phosphotyrosine. Waved-2 mice, which have impaired EGFR kinase activity, exhibited less mammary development than did wild-type (wt) mice when both were evaluated at 36 days of age. Because EGFR knockout (KO) mice die shortly after birth, glands from the newborns were implanted under the renal capsules of female nude mice. Under these conditions, extensive ductal growth was observed in mammary glands from wt animals; in contrast, glands from EGFR KO mice failed to grow beyond rudimentary structures. Tissue recombinants revealed that the wt fat pad supported the morphogenesis of EGFR KO epithelium, whereas the EGFR KO fat pad did not. Taken together, these data suggest that EGFR is essential for morphogenesis of the mammary ducts and functions during this period of mammary development as a heterodimer with erbB-2 in the mammary stroma.