Targeting drugs to receptors involved in tumor angiogenesis is a novel and promising approach to improve cancer treatment. In this study, we evaluated the antitumor activity of paclitaxel (PTX) conjugated with a bicyclic peptide E[c(RGDyK)](2) (RGD) in a metastatic breast cancer cell line (MDA-MB-435). The cyclic RGD peptide selectively binds to alpha(v) integrin receptors that are highly expressed in metastatic cancer cells. PTX, an antimicrotubule agent, is a potent antitumor agent commonly used in the treatment of advanced metastatic breast cancer. The in vitro results showed that RGD peptide inhibited cell cycle proliferation by arresting cells in G(0)/G(1)-phase. The PTX-RGD conjugate inhibited cell proliferation with activity comparable to that observed for paclitaxel, both of which were mediated by an arrest of G(2)/M-phase of the cell cycle followed by apoptosis. Although the PTX-RGD conjugate showed slightly decreased integrin binding affinity than the unconjugated peptide, it indicated integrin specific accumulation in vivo. (125)I-Labeled PTX-RGD showed highest tumor uptake at 2 h postinjection (2.72 +/-0.16%ID/g) and best tumor/background contrast after 4 h postinjection. Our results demonstrate the potential of tumor-targeted delivery of paclitaxel based on the specific recognition of cell adhesion molecule alpha(v)beta(3) integrin to reduce toxicity and enhance selective killing of cancer cells.