In vivo knowledge of the spatial distribution of viable, necrotic, and hypoxic areas can provide prognostic information about the risk of developing metastases and regional radiation sensitivity and may be used potentially for localized dose escalation in radiation treatment. In this study, multimodality in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging using stereotactic fiduciary markers in the Dunning R3327-AT prostate tumor were performed, focusing on the relationship between dynamic contrast-enhanced (DCE) MRI using Magnevist (Gd-DTPA) and dynamic (18)F-fluoromisonidazole ((18)F-Fmiso) PET. The noninvasive measurements were verified using tumor tissue sections stained for hematoxylin/eosin and pimonidazole. To further validate the relationship between (18)F-Fmiso and pimonidazole uptake, (18)F digital autoradiography was performed on a selected tumor and compared with the corresponding pimonidazole-stained slices. The comparison of Akep values (kep = rate constant of movement of Gd-DTPA between the interstitial space and plasma and A = amplitude in the two-compartment model (Hoffmann U, Brix G, Knopp MV, Hess T and Lorenz WJ (1995). Magn Reson Med 33, 506-514) derived from DCE-MRI studies and from early (18)F-Fmiso uptake PET studies showed that tumor vasculature is a major determinant of early (18)F-Fmiso uptake. A negative correlation between the spatial map of Akep and the slope map of late (last 1 hour of the dynamic PET scan) (18)F-Fmiso uptake was observed. The relationships between DCE-MRI and hematoxylin/eosin slices and between (18)F-Fmiso PET and pimonidazole slices confirm the validity of MRI/PET measurements to image the tumor microenvironment and to identify regions of tumor necrosis, hypoxia, and well-perfused tissue.