IGF-I and epidermal growth factor (EGF) stimulate both normal mammary epithelial cell (MEC) growth as well as tumorigenesis. While both growth factors increase DNA synthesis in MEC, how they evoke a greater response in combination when they activate similar signaling pathways remains unknown. Presently, we investigated the signaling pathways by which these mitogens act in concert to increase DNA synthesis. Only EGF activated the MAPK pathway, and no further increase in MAPK activation was observed when both mitogens were added together. Both growth factors activated the phosphatidylinositol-3 kinase (PI3K) pathway, and simultaneous treatment enhanced phosphorylation of both AKT and its downstream target, p70S6K. The enhanced activation of AKT was observed at multiple time points (5 and 15 min) and growth factor concentrations (2.5 to 100 ng/ml). IGF-I activated AKT via IRS-1 and p85, the regulatory subunit of PI3K. Treatment with EGF had no effect on IRS-1; however, it activated the EGF receptor, SHC, and c-Src. EGF treatment caused the association of SHC with Grb2, and of Gab2 with phospho-SHC phospho-Gab1, Grb2, and p85. Interestingly, inhibition of Src activation blocked the ability of EGF, but not IGF-I, to activate AKT. This corresponded with a decrease in phosphorylation of the EGF receptor and its association with phospho-SHC, as well as downstream signaling. Unexpectedly, inhibition of Src increased basal MAPK activation. This is the first study to show that EGF and IGF-I use separate upstream components within a given MEC line to enhance AKT phosphorylation, contributing to increased DNA synthesis.