The von Hippel-Lindau gene product (pVHL) interacts with and inhibits the cellular transcription factor elongin. However, the subcellular localization of pVHL has remained uncertain. Naturally occurring pVHL mutants which fail to interact with elongin have been described in patients with VHL disease or sporadic renal cell carcinoma (RCC). Here, we have examined the cellular expression pattern of endogenous pVHL in different RCC cell lines by immunocytochemistry and confocal microscopy. Both anti-N-terminal and anti-C-terminal pVHL antibodies were able to recognize endogenous wild-type pVHL expressed by the RCC cells studied. A C-terminal truncated VHL mutant expressed by RCC cell line A498 was detected only by the N-terminal antibody but not by the C-terminal antibody as expected. The overall staining patterns of these cell lines are similar, with a predominant nuclear speckled pattern and a moderate cytoplasmic staining in subconfluent cell cultures. Interestingly, when cells reached confluency, more prominent nuclear staining with little or no cytoplasmic expression was observed. By using double labeling with anti-VHL and anti-bromodeoxyuridine (BrdU) antibodies and cell cycle analyses, we found that in the G1/G0-phase, pVHL was localized exclusively in the nucleus associated with distinctive large subnuclear structures, whereas the majority of the cells in S-phase of the cell cycle also showed a diffuse cytoplasmic staining. Our results indicate that subcellular localization of pVHL is regulated in a cell cycle-dependent manner.