Hypoxia is an important resistance factor in radiotherapy and measuring its spatial distribution in tumors non-invasively is therefore of major importance. This study characterizes the hypoxic conditions of three tumor sublines (AT1, HI and H) of the Dunning R3327 prostate tumor model, which differ in histology, differentiation degree, volume doubling time and androgenic sensitivity, using dynamic Fluoromisonidazole ((18)F-FMISO)-Positron Emission Tomography/Computed Tomography (PET-CT) and histology. Measurements were performed for two tumor volumes (average 0.8±0.5 cm(3) vs 4.4±2.8 cm(3)). Data were analyzed according to tumor subline as well as to the shape of the time activity curves (TACs), based on standardized uptake values (SUVs) and a two-tissue compartment model. Quantitative immunohistochemical studies of the hypoxic fraction, vessel density and vessel size were performed using pimonidazole, Hoechst 33342 and CD31 dyes. No significant FMISO uptake was found in small tumors, which had a mean SUV of 0.64±0.36, 0.55±0.10 and 0.45±0.08, for AT1, HI and H sublines respectively. In large tumors, the SUVs were 1.33±0.52, 1.12±0.83 and 0.63±0.16 for AT1, HI and H sublines and the corresponding hypoxic fractions obtained with pimonidazole staining were 0.62±0.23, 0.54±0.24 and 0.07±0.10, respectively. The AT1- was the most and H-tumor was the least hypoxic for both methods (P<0.05). All measurements were able to discriminate different hypoxic conditions, however despite SUV and kinetic parameters correlated with the three identified TAC shapes, most of the histological results did not. These results demonstrate impact and limitations of static and dynamic PET-CT measurements to assess hypoxia non-invasively.