Longitudinal studies of lactate MR spectroscopic imaging (MRSI) and dynamic contrast-enhanced MR imaging (DCE-MRI) were performed at 4.7 T in two prostate tumor models grown in rats, the Dunning R3327-AT (AT) and Dunning H (H), to determine the potential of lactate and the perfusion/permeability parameter Akep as markers of tumor aggressiveness. Subcutaneous AT (n = 12) and H (n = 6) tumors were studied at different volumes between 100 and 2900 mm3 (Groups 1 to 5). Lactate concentration was determined from Selective Multiple Quantum Coherence (Sel-MQC) MRSI using phantom substitution method. Tumor enhancement after administration of Gd-DTPA was analyzed using the Brix-Hoffmann model and the Akep parameter was used as a measure of tumor perfusion/permeability. Lactate was not detected in the smallest AT tumors (Group 1; 100–270 mm3). In larger AT tumors, lactate concentration increased from 2.8 ± 1.0 mM (Group 2; 290–700 mm3) to 8.4 ± 2.9 mM (Group 3; 1000–1340 mm3), 8.2 ± 2.2 mM (Group 4; 1380–1750 mm3), and then decreased to 5.0 ± 1.7 mM (Group 5; 1900–2500 mm3) and was consistently higher in the tumor core than in the rim. Lactate was not detected in any of the Dunning H tumors. The mean tumor Akep values decreased with increasing volume in both tumor types, but were significantly higher in H tumors. In AT tumors, the Akep values were significantly higher in the rim than in the core. Histological hypoxic and necrotic fractions in AT tumors increased with volume from 0% in Group 1 to about 20% and 30%, respectively, in Group 5. Minimal amounts of hypoxia and necrosis were found in H tumors of all sizes. Thus the presence of lactate and heterogeneous perfusion/permeability are signatures of aggressive, metabolically deprived Dunning R3327-AT tumor, but not the slow-growing Dunning H tumor.