One of the major targets for breast cancer therapy is the epidermal growth factor receptor (EGFR) and related receptors, which signal via different signal transduction pathways including the mitogen-activated protein kinase (MAPK) pathway. This study determined whether there is a correlation between EGFR/HER2 status and MAPK (ERK1/2) phosphorylation in breast cancer cells, and how this affects the response to an inhibitor of the receptors. Expression of EGFR, HER2 and phosphorylated ERK1/2 were measured by immunoblotting in a panel of breast cancer cell lines. Several lines expressed high levels of pERK1/2, with no obvious correlation with the level of EGFR/HER2. The EGFR tyrosine kinase inhibitor PKI166 inhibited growth and induced apoptosis in some cells with high levels of growth factor receptors (MDA-MB-468, SUM149, SKBR3), but was less effective in cells that also had high basal ERK1/2 activity (MDA-MB-231). The combination of an inhibitor of MAPK signalling (U0126) and PKI166 produced significantly more inhibition and apoptosis than either agent alone. This suggests that constitutive activation of the MAPK pathway may bypass inhibition of EGFR/HER2 tyrosine kinases, and lead to insensitivity to agents targeting the receptors. However, inhibiting both EGFR/HER2 and MAPK signalling can result in significant growth inhibition and apoptosis of EGFR-expressing breast cancer cells.