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Tumor heterogeneity complicates the quantification of tumor microvascular characteristics assessed by dynamic contrast-enhanced MRI (DCE-MRI). To address this issue a novel approach was developed that combines DCE-MRI with diffusion-based multispectral (MS) analysis to quantify the microvascular characteristics of specific tumor tissue populations. Diffusion-based MS segmentation (feature space: apparent diffusion coefficient,T2 and proton density) was performed to identify tumor tissue populations and the DCE-MRI characteristics were determined for each tissue class. The ability of this MS DCE-MRI technique to detect microvascular changes due to treatment with an antibody (G6-31) to vascular endothelial growth factor-A (VEGF) was evaluated in a tumor xenograft mouse model. Anti-VEGF treatment resulted in a significant reduction in Ktrans for the MS viable tumor tissue class (−0.0034 ± 0.0022 min−1,P< 0.01) at 24 hr posttreatment that differ significantly from the change observed in the control group (0.0002 ± 0.0025 min−1). Viable tumor Ktrans for the anti-VEGF group was also reduced 62% relative to the pretreatment values (P< 0.01). Necrotic tissue classes were found to add only noise to DCE-MRI estimates. This approach provides a means to measure physiological parameters within the viable tumor and address the issue of tumor heterogeneity that complicates DCE-MRI analysis. Magn Reson Med, 2008. © 2008 Wiley-Liss, Inc.