Objectives-Actively targeting ultrasound contrast agents to tumor vasculature improves contrast-enhanced sonography of tumor angiogenesis. This report summarizes an evaluation of multitargeted microbubbles, comparing single-, dual-, and tripletargeted motifs. Methods-Microbubbles were avidin-biotin linked to antibodies against mouse αVβ 3- integrin, P-selectin, and vascular endothelial growth factor receptor 2. These receptors are constitutively overexpressed in tumor vasculature. Binding comparisons between targeted microbubble groups were evaluated on mouse SVR angiosarcoma endothelial cells. Levels of the targeted receptors were characterized with flow cytometry. Targeted microbubble groups were administered to human MDA-MB-231 breast cancer tumor-bearing mice (n = 3) followed by contrast-enhanced sonography in a microbubble-sensitive harmonic imaging mode implemented on an ultrasound scanner equipped with a linear array transducer (5 MHz transmit and 10 MHz receive) to evaluate differences in microbubble accumulation in the tumor vasculature. Results-In vitro analysis showed a 50% increase (P < .001) in triple-targeted microbubble binding over dual-targeted microbubble groups in mouse SVR cells. Mice bearing MDA-MB-231 tumors showed a 40% increase in tumor image intensity after dosing with triple-targeted microbubbles compared with single- and dual-targeted microbubbles (P = .006). Histologic staining confirmed the presence of αVβ3-integrin, Pselectin, and vascular endothelial growth factor receptor 2 in the tumors. Conclusions-Microbubble accumulation in the tumor vasculature was improved using a triple-targeted microbubble approach. © 2011 by the American Institute of Ultrasound in Medicine.