Renal-cell carcinoma (RCC) is the most common neoplasm of the kidney, accounting for about 3% of all adult malignancies. Histopathologically, 80% of all cases can be classified as clear-cell RCC. Of these, approximately 55% to 70% are associated with mutations in the von Hippel-Lindau (VHL) tumor-suppressor gene. Here, new mutations of the VHL gene were defined by the use of temperature gradient gel electrophoresis and subsequent sequencing. In addition, DNA hypermethylation, an alternative mechanism of VHL gene silencing, was evaluated by methylation-specific PCR. Twenty-six clear-cell, 3 chromophilic, and 2 chromophobic RCC cell lines were analyzed. Among the clear-cell RCC cell lines tested, 12 (47%) contained 13 mutations overall: 8 (62%) in exon 1, 3 (23%) in exon 2, and 2 (15%) in exon 3. Ten of these mutations have thus far not been described. All single base pair changes were transversions. Six mutations led to alteration of a single amino acid. Seven mutations generated a frameshift or a stop codon. One cell line contained a complex duplication of 36 bp. All cell lines with mutations showed loss of heterozygosity in the VHL gene. No mutations could be detected in the chromophilic or chromophobic RCC samples. Significant hypermethylation was not observed in any of the cell lines. These data provide further evidence that distinct mutations in the VHL gene are a characteristic feature of clear-cell RCC. In contrast, hypermethylation of the gene is probably a rare event. The high frequency of transversion mutations suggests a role for exogenous carcinogens in the etiology of clear-cell RCCs.