Germ-line mutations in the human MSH2 (hMSH2) gene account for about 40% of known defects in kindreds with hereditary nonpolyposis colon cancer. We describe a simple fusion protein assay for detection of hMSH2 nonsense mutations in yeast. Detection of nonsense mutations with this assay is severely compromised in many cases by nonsense-mediated mRNA decay, a physiological process that destabilizes the mutant RNA. Triggering of nonsense-mediated decay requires mRNA scanning by the ribosome to detect the stop codon. We show that treatment of cells with the translation inhibitor puromycin suppresses nonsense-mediated decay and facilitates the detection of nonsense mutations in clinical samples by cDNA sequencing, in vitro protein truncation tests, and the yeast fusion protein assay. Given the prevalence of chain-terminating mutations in human disease genes, puromycin treatment of blood samples should improve the signal-to-noise ratio and hence the sensitivity of many RNA-based diagnostic tests. Paradoxically, the yeast hMSH2::ADE2 fusion protein assay also detects some in-frame mutations, presumably through an effect on the folding of the fusion protein.