In the Eker rat model, inactivation of the Tuberous Sclerosis-2 (Tsc-2) tumor suppressor gene leads to high frequency of spontaneous renal cell carcinoma (RCC). By analogy to human RCC in which mutations in the von Hippel-Lindau (VHL) tumor suppressor gene result in accumulation of hypoxia-inducible factor alpha (HIFalpha) and up-regulation of vascular endothelial growth factor (VEGF), we investigated the regulation of HIF and its target gene VEGF in rat RCC resulting from Tsc-2 defects. To examine HIFalpha activity, a panel of rat renal epithelial cells were analyzed for expression of HIF1alpha and the homologous protein, HIF2alpha, under normoxic and hypoxic conditions. RCC-derived cell lines exhibited high basal levels of HIF activity as determined using hypoxia response element-luciferase reporter constructs. HIF2alpha was stabilized in RCC-derived cell lines and in five of six primary tumors compared with normal kidney, which was consistent with the high levels of hypoxia response element-reporter activity observed in the cell lines. Primary RCCs that developed in Eker rats were highly vascularized, which was similar to their human counterparts. Furthermore, reverse-transcriptase PCR and immunoblotting demonstrated that VEGF was abundantly expressed in both rat RCC cell lines and primary tumors. The 120-, 164-, and 188-amino-acid isoforms of VEGF were expressed at the RNA and protein levels in RCC-derived cell lines, although only a single band was observed in primary tumors. Taken together, these data suggest that RCC caused by loss of the Tsc-2 tumor suppressor gene (which retain wild-type Vhl) up-regulate VEGF via a HIF2alpha-mediated mechanism. Thus, loss of Tsc-2 and VHL tumor suppressor gene function appears to have similar consequences in Eker rats and humans respectively, identifying dysregulation of HIFalpha and VEGF expression as a common pathway for the development of RCC in different species and in tumors with different molecular etiologies.