RATIONALE AND OBJECTIVES: Non-invasive longitudinal imaging of tumor vasculature could provide new insights into the development of solid tumors, facilitating efficient delivery of therapeutics. In this study, we report three-dimensional imaging and characterization of tumor vascular architecture using a nanoparticle contrast agent and high-resolution computed tomography (CT) imaging. MATERIALS AND METHODS: Five Balb/c mice implanted with 4T1/Luc syngeneic breast tumors cells were used for the study. The nanoparticle contrast agent was systemically administered and longitudinal CT imaging was performed pre-contrast and at serial time points post-contrast, for up to 7 days for studying the characteristics of tumor-associated blood vessels. Gene expression of tumor angiogenic biomarkers was measured using quantitative real-time polymerase chain reaction. RESULTS: Early-phase imaging demonstrated the presence of co-opted and newly developed tumor vessels. The co-opted vessels demonstrated wall-permeability and "leakiness" characteristics evident by an increase in extravascular nanoparticle-based signal enhancement visible well beyond the margins of tumor. Diameters of tumor-associated vessels were larger than the contralateral normal vessels. Delayed-phase imaging also demonstrated significant accumulation of nanoparticle contrast agent both within and in areas surrounding the tumor. A heterogeneous pattern of signal enhancement was observed both within and among individual tumors. Gene-expression profiling demonstrated significant variability in several angiogenic biomarkers both within and among individual tumors. CONCLUSIONS: The nanoparticle contrast agent and high-resolution CT imaging facilitated visualization of co-opted and newly developed tumors vessels as well as imaging of nanoparticle accumulation within tumors. The use of this agent could provide novel insights into tumor vascular biology and could have implications on the monitoring of tumor status.