Mutations in the hepatocyte nuclear factor-1alpha (HNF-1a) gene cause maturity-onset diabetes of the young (MODY). Approximately 30% of these mutations generate mRNA transcripts harboring premature termination codons (PTCs). Degradation of such transcripts by the nonsense-mediated decay (NMD) pathway has been reported for many genes. To determine whether PTC mutant transcripts of the HNF-1alpha gene elicit NMD, we have developed a novel quantitative RT-PCR assay. We performed quantification of ectopically expressed mutant transcripts relative to normal transcripts in lymphoblastoid cell lines using a coding single nucleotide polymorphism (cSNP) as a marker. The nonsense mutations R171X, I414G415ATCG-->CCA, and P291fsinsC showed reduced mutant mRNA expression to 40% (P = 0.009), <0.01% (P </= 0.0001), and 6% (P = 0.001), respectively, of the normal allele. Transcript levels were restored using the translation inhibitor cycloheximide, indicating that the instability arises from NMD. The missense mutations G207D and R229P did not show NMD although R229P exhibited moderate RNA instability. This study provides the first evidence that HNF-1alpha PTC mutations may be subject to NMD. Mutations that result in significant reduction of protein levels due to NMD will not have dominant-negative activity in vivo. Haploinsufficiency is therefore likely to be the most important mutational mechanism of HNF-1alpha mutations causing MODY.