The alpha(v)beta(3) integrin is overexpressed on proliferating endothelial cells such as those present in growing tumors as well as on tumor cells of various origins. Tumor-induced angiogenesis can be inhibited in vivo by antagonizing the alpha(v)beta(3) integrin with small peptides containing the arginyl-glycyl-aspartic acid (RGD) amino acid sequence. The divalent cyclic peptide E-[c(RGDfK)(2)] is a novel ligand-based vascular-targeting agent that binds integrin alpha(v)beta(3) and demonstrated high uptake in OVCAR-3 xenograft tumors. In this work, we coupled the 2'-OH-group of paclitaxel through an aliphatic ester to the amino group of E-[c(RGDfK)(2)] or the control peptide c(RADfK), thus obtaining the derivatives E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel. Subsequently, we investigated the activity of the paclitaxel derivatives using several well-established in vitro angiogenesis assays: using a standard 72 h endothelial cell proliferation assay, we showed that both E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) in a similar manner as free paclitaxel (IC(50) value approximately 0.4 nM), an observation that can be explained by the half-life of the paclitaxel ester bond in the conjugates of approximately 2h at pH 7. In contrast, a 30-min exposure of the cells to the three drugs showed a clear difference between free paclitaxel, E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel with IC(50) values of 10nM, 25 nM, and 60 nM, respectively. These differences are very likely due to the different routes of cellular entry of these three molecules. While the hydrophobic paclitaxel diffuses rapidly through the cell membrane, the charged peptide-containing derivative E-[c(RGDfK)(2)]-paclitaxel binds to the overexpressed alpha(v)beta(3) integrin in order to enter the cells via receptor-mediated endocytosis. The differences between the derivatives were further demonstrated using an endothelial cell adhesion assay. Inhibition of cell attachment was observed only with the E-[c(RGDfK)(2)]-paclitaxel derivative indicating its specificity to the growing endothelial cells. Furthermore, E-[c(RGDfK)(2)]-paclitaxel inhibited both endothelial cells migration and capillary-like tube formation. These results further demonstrate their antiangiogenic properties. In vivo studies in an OVCAR-3 xenograft model demonstrated no antitumor efficacy for either E-[c(RGDfK)(2)] or E-[c(RGDfK)(2)]-paclitaxel compared to moderate efficacy for paclitaxel.