In order to evaluate the sensitivity and specificity of the recently introduced high-throughput method DHPLC (denaturing high performance liquid chromatography) for mutation screening in the VHL tumor suppressor gene, we subjected DNA from 43 unrelated VHL patients with previously sequenced VHL germline mutations to this method. In addition, 36 genomic DNAs of unrelated individuals suspected of being VHL carriers but with unknown germline status were analyzed by DHPLC and sequencing. The aims of the present study were to compare mutation results obtained by direct sequencing and DHPLC, and a comparison of two different DHPLC systems. The sensitivity of DHPLC was tested with two commercial devices and protocols, i.e., the Varian-Helix system and the Wave Nucleic Acid Fragment Analysis system. Both resolved all but one mutation in exons 2 and 3 of the VHL gene. In contrast, the GC-rich exon 1 showed discrepancies in the rate of mutation detection. Whereas the Varian-Helix system detected 10/15 (67%) of the known mutations, the Wave Nucleic Acid Fragment Analysis system detected 13/14 (93%). All three mutations in samples with unknown mutation status were revealed by both systems raising the mutation detection rate to 72% and 94%, respectively. Cases with different substitutions at the same nucleotide showed different elution profiles, but similar elution profiles could be obtained from different mutations. The Wave Nucleic Acid Fragment Analysis system detected most VHL mutations; however, when a 100% detection rate is needed, sequencing is still required and must therefore be the standard VHL mutation detection procedure. Once a family-specific mutation has been established, DHPLC may be suitable for the rapid and cost-effective determination of VHL carrier status in family members.