Deletions of one or more exons in the mismatch repair genes MLH1 and MSH2 have been implicated in a significant fraction of hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Multiplex ligation-dependent probe amplification (MLPA) detection of deletions of multiple sequential exons is widely accepted; however, there is concern over the reliability of MLPA results showing single exon deletions. Given the clinical implications of a diagnosis of Lynch syndrome, it is important to use an alternative technique to confirm single exon deletions. To verify single exon deletions, we developed a SYBR Green-based quantitative polymerase chain reaction (PCR) assay. Clinical DNA samples containing deletions in 33 of the 35 exons in MLH1 and MSH2, previously screened by MLPA, were evaluated by quantitative PCR. Gene dosage ratios were determined by both the relative standard curve method and by the 2(-DeltaDeltaC(T)) method. Deleted exons had gene dosage ratios of 0.4 to 0.6, while nondeleted exons exhibited ratios of 0.8-1.3. We found 100% concordance between the quantitative PCR and MLPA results, including confirmation of all single exon deletions. The 2(-DeltaDeltaC(T)) method was as accurate as using standard curves for the calculation of ratios. Single exon deletions in MLH1 and MSH2 can be verified using quantitative PCR. Assays using this method are simple to design and easy to perform, making them ideal for confirmatory testing.