We have previously demonstrated that exposure to genistein early in life protects against chemically-induced mammary cancer in rats. To gain insight into the mechanism of action, we have investigated the expression of the EGF-signaling pathway in the mammary glands of 21 and 50 day old rats treated on days 16, 18, and 20 postpartum with 500 microg genistein/g body weight (B.W.) or an equivalent volume of the vehicle, dimethylsulfoxide (DMSO). This prepubertal genistein treatment up-regulated TGF-alpha and the EGF-receptor (EGFR), but not EGF, in mammary terminal ductal structures at day 21 postpartum. TGF-alpha, EGF and EGFR mRNA levels were similar in 21 day old control- and genistein-treated animals. At day 50 postpartum, mammary glands of genistein treated rats had more lobules and fewer terminal end buds (TEBs) and terminal ducts (TDs), i.e. they were more differentiated. TGF-alpha mRNA levels were down-regulated in TEB of proestrous and estrous females; EGF mRNA levels were down-regulated in TDs of proestrous, but not in estrous females; and EGFR mRNA levels were not altered in 50 day old proestrous or estrous female rats. EGFR immunostaining intensity was decreased in TEBs, but not in the total gland. EGF was increased in TEBs and TDs. TGF-alpha, EGF and EGFR were also observed in the stroma and fat pad, but genistein treatment did not alter the expression of these proteins in those locations. TGF-alpha, but not EGF and EGFR, immunostaining was observed in cell nuclei (not modulated by genistein), suggesting that this growth factor may act directly on nuclear events such as transcription and DNA replication. For comparative purposes, prepubertal diethylstilbestrol treatment was investigated and found to decrease EGFR immunostaining intensity and total IHC staining in all terminal ductal structures. We conclude that prepubertal genistein treatment directly stimulates TGF-alpha and EGFR to enhance mammary gland differentiation. This programs the differentiated cells for a down-regulated EGF-signaling pathway in TEBs and TDs of adult mammary glands. Reduced EGFR expression at time of carcinogen exposure may account for genistein programming against mammary cancer.