Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody directed against HER-2. The objective response rate to trastuzumab monotherapy is 12?34% for a median duration of 9 months, by which point most patients become resistant to treatment. We created two trastuzumab-resistant (TR) pools from the SKBR3 HER-2-overexpressing breast cancer cell line to study the mechanisms by which breast cancer cells escape trastuzumab-mediated growth inhibition. Both pools maintained her-2 gene amplification and protein overexpression. Resistant cells demonstrated a higher S-phase fraction by flow cytometry and a faster doubling time of 24?36 h compared with 72 h for parental cells. The cyclin-dependent kinase inhibitor p27kip1 was decreased in TR cells, and cyclin-dependent kinase 2 activity was increased. Importantly, exogenous addition of p27kip1 increased trastuzumab sensitivity. Additionally, resistant cells displayed heightened sensitivity to the proteasome inhibitor MG132, which induced p27kip1 expression. Thus, we propose that trastuzumab resistance may be associated with decreased p27kip1 levels and may be susceptible to treatments that induce p27kip1 expression.