Previously, our laboratory has shown that prolactin (PRL) inhibits epidermal growth factor (EGF)-induced DNA synthesis. One pathway for the initiation of DNA synthesis is EGF-receptor (EGF-R) signaling through Ras and mitogen-activated protein kinase (MAPK). To determine the effects of PRL on EGF-induced MAPK activation and phosphorylation, MAPK or phosphotyrosine (Tyr(P)) was immunoprecipitated from normal murine mammary epithelial (NMuMG) cells treated with PRL (100 ng/ml) and/or EGF (10 ng/ml) for 10-min periods. EGF-induced phosphorylation and activation were then examined by Western analysis and a myelin basic protein (MBP)-specific kinase assay. The p42 isoform of MAPK showed a distinct decrease in activity and phosphorylation when cells were treated with PRL. Concluding that PRL affects EGF signaling upstream of MAPK, we examined the effect of PRL on EGF-induced Ras activity. NMuMG cells were incubated with [32P]orthophosphoric acid, treated as described above, immunoprecipitated with an antibody specific to Ras, and nucleotides were eluted and separated by TLC. Ras activity as measured by GTP:GDP ratio was increased by EGF, but not by PRL. Additionally, PRL in combination with EGF abolished the ability of EGF to induce Ras activity. Those studies suggest that PRL alters the EGF signaling pathway upstream of Ras. Because Ras activation by EGF involves EGF-stimulated association of EGF-R with Grb2, the EGF-R was immunoprecipitated and a Western blot was probed for Grb2. As expected we found that EGF stimulated an association of EGF-R with Grb2, PRL, however, blocked this association. When we looked at the ability of Shc to associate with the EGF-R, we found that PRL and EGF had little effect on this association. The studies demonstrate that PRL either directly or indirectly inhibits the ability of EGF to induce EGF-R association with Grb2, to activate Ras, and to activate and phosphorylate MAPK.