We present a novel method for accurate quantification of single nucleotide polymorphism (SNP) variants in transcripts and pooled DNAs in a one-tube reaction. Our approach is based on singlenucleotide primer extension (SNuPE) and laser-induced fluorescence capillary electrophoresis (LIF-CE), and takes advantage of distinct mobilities of SNuPE products with different nucleotides incorporated at their 3¢ ends. The method, called SNuPE-ONCE, was tested on two polymorphisms and five mutations that comprised the three most frequent (~70%) nucleotide changes in the human genome (C/T, A/G, and A/T). The usefulness of the method was demonstrated by analyzing nonsense-mediated mRNA instability in fibroblasts. Our data show 1) that the method provides highly reproducible relative allele frequencies (SD<0.017) with a good accuracy (e.g. for heterozygotes 0.500 ± 0.036, P=0.01), depending on the sequence and the proportion of the SNP variants in the sample, and 2) that relative allele frequencies as low as 1% can be detected quantitatively and unambiguously. Our assay relies on a CE instrument available in many laboratories and offers a useful method for quantitative SNP genotyping as well as for a variety of expression studies. Hum Mutat 19:5868, 2002.