Abstract Objectives In tumors, hypoxia stimulates angiogenesis and correlates with treatment resistance and poor prognosis. We have previously demonstrated hypoxia in human renal cell carcinoma (RCC) via direct oxygen probe measurements. Carbonic anhydrase IX (CA IX) is a protein stimulated by hypoxia and involved in angiogenesis, and is a potential tumor target for imaging and therapies using cG250, a monoclonal antibody that recognizes CAIX. Our objectives were to characterize intratumoral hypoxia in a human RCC xenograft model using oxygen probe measurements; investigate if 124 I-cG250 targets RCC correlating uptake on noninvasive positron emission tomography-computerized tomography (PET-CT) against traditional biodistribution studies, and investigate CAIX expression in this RCC model. Methods BALB/c nude mice had human RCC (SK-RC-52) subcutaneously xenografted with oxygen levels measured by probe. Positron emission tomography (PET/CT) and biodistribution studies ( 124 I-cG250) were correlated with oxygen measurements. Immunohistochemistry and autoradiography were performed on selected tumors to confirm CAIX expression Results Oxygen tension in normal tissue (muscle) was 35.08 ± 2.41 mmHg (mean ± 95% CI), significantly greater compared to xenograft SK-RC-52 tumors at 5.02 ± 1.12 mmHg. Biodistribution studies of 124 I-cG250 demonstrated isotope uptake in SK-RC-52 xenografts peaking at 23.45 ± 5.07% ID/g (mean ± SD) 48 hours after antibody injection, which was maintained for a further 2 days (19.43 ± 4.31 and 10.64 ± 5.64 % ID/g, respectively). PET studies demonstrated excellent localization of 124 I-cG250 in tumor, and a significant correlation between SUVmean, SUVmax, and %/ID 124 I-cG250. CAIX expression was present in all groups studied but there was no significant correlation between it and any oxygen parameter studied. Conclusion Intratumoral hypoxia does exist within a human RCC xenograft model using invasive oxygen probe measurements. 124 I-cG250 targets RCC with correlation between uptake on noninvasive PET-CT studies and traditional biodistribution studies opening the possibility of using PET/CT in future studies. Finally, CAIX expression was not related to hypoxia in this model, supporting the hypothesis that cell lines may subvert known hypoxia mechanisms in hypoxic environments.