Human epidermal growth factor receptor-2 (HER2) and epidermal growth factor receptor (EGFR) heterodimerize to activate mitogenic signaling pathways. We have shown previously, using MCF7 subcloned cell lines with graded levels of HER2 expression, that responsiveness to trastuzumab and AG1478 (an anti-EGFR agent), varied directly with levels of HER2 expression. HER2 and EGFR up-regulate vascular endothelial growth factor (VEGF), a growth factor that promotes angiogenesis and participates in autocrine growth-stimulatory pathways that might be active in vitro. Here, we show that trastuzumab, erlotinib, and bevacizumab, individually and in combination, inhibit cell proliferation in a panel of unrelated human breast cancer cell lines, in proportion to their levels of HER2 expression. The combination of all three drugs provided a greater suppression of growth than any single drug or two-drug combination in the high HER2-expressing cell lines (P < 0.001). Combination index analysis suggested that the effects of these drugs in combination were additive. The pretreatment net level of VEGF production in each cell line was correlated with the level of HER2 expression (r = 0.883, P = 0.016). Trastuzumab and erlotinib each reduced total net VEGF production in all cell lines. Multiparameter flow cytometry studies indicated that erlotinib alone and the triple drug combination produced a prolonged but reversible blockade of cells in G1, but did not increase apoptosis substantially. These studies suggest that the effects of two and three-drug combinations of trastuzumab, erlotinib, and bevacizumab might offer potential therapeutic advantages in HER2-overexpressing breast cancers, although these effects are of low magnitude, and are likely to be transient.