Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G₁ arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27^Kip1, and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27^Kip1 onto Cdk2 complexes, an event preceding increased p27^Kip1 expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27^Kip1sequestration) and the loss of p27^Kip1 from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27^Kip1 redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27^Kip1 protein levels remained constant. Antisense-mediated inhibition of p27^Kip1 expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27^Kip1 accumulation, p27^Kip1 redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G₁ block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27^Kip1 sequestration proteins, thus deregulating the G₁/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.