Epidermal growth factor receptor (EGFR) and HER-2/ErbB2 are members of the Erb family of signaling receptors. ErbB2 is overexpressed in many different cancers and has been linked to enhanced malignancy of tumors. We have examined the cellular translocation of Raf-1 during EGF-dependent signal transduction in two breast tumor cell lines, BT20 and SKBR3. Treatment of BT-20 breast cancer cells, which express EGFR, with EGF resulted in rapid (5 minutes) accumulation of EGFR and Raf-1 into plasma membrane-associated endocytotic vesicles. However, at later time points (30 minutes) only EGFR was endocytosed and Raf-1 dissociated from the plasma membrane and was found in the cytosol. In SKBR3 breast cancer cells, which express high levels of EGFR and ErbB2, treatment with EGF also resulted in rapid accumulation of EGFR and Raf-1 into endocytotic vesicles, but EGFR endocytosis was inhibited and Raf-1 remained associated with the plasma membrane for a prolonged period. The role of ErbB2 in the retention of Raf-1 at the plasma membrane was confirmed in BT-20 cells transfected with ErbB2. BT-20 cells expressing ErbB2 and treated with EGF retained Raf-1 at the plasma membrane for prolonged periods, whereas Raf-1 rapidly dissociated from the plasma membrane in EGF-stimulated cells transfected with a control vector. The presence of Raf-1 at the plasma membrane correlated with activation of Raf-1 and MAP kinase. Cells that expressed ErbB2 and treated with EGF showed prolonged activation of Raf-1 and MAP kinase compared with cells that expressed low levels of ErbB2. These results suggest that expression of ErbB2 promoted retention of Raf-1 in the plasma membrane, resulting in prolonged activation of the MAP kinase cascade, which may contribute to enhanced malignancy in ErbB2-expressing cancers.