Cytochrome P450CYP2A6 (CYP2A6) is the predominant enzyme responsible for the metabolism of nicotine to cotinine. Two variants have been identified that encode products presumed to have little or no activity. A previous study suggested that carriers of at least one copy of either null variant may be protected against tobacco dependence, while tobacco-dependent carriers smoke fewer cigarettes. However, different laboratories have reported widely disparate CYP2A6 allele frequencies across European populations. These differences prompted us to reexamine the genotyping methods for CYP2A6. We developed an improved genotyping strategy using CYP2A6-specific nested PCR, and differential restriction enzyme digestion to identify variant nucleotides in exon 3. We used sequencing to verify genotype results and to assess the sequence of exon 4, which previous work predicted should correspond to "wild-type" CYP2A6 sequence. In addition, we developed a new nomenclature in which CYP2A6*1 is designated CYP2A6*A1-*B1, CYP2A6*2 is CYP2A6*A2, and CYP2A6*3 is CYP2A6*B2. The frequencies of CYP2A6*A2 and CYP2A6*B2 were then estimated in samples from six populations. Sequencing confirmed CYP2A6*A2 genotypes in all cases. Unexpectedly, sequencing demonstrated exon 4 sequence corresponding to CYP2A7 in samples genotyped as CYP2A6*B2. In the population study, we found consistently low allele frequencies (</=5%) for CYP2A6*A2 and CYP2A6*B2 in all samples examined. The molecular data from this study suggest that further physical mapping may be necessary to clarify the structure of CYP2A6*B2. The population results suggest that in many populations the frequencies of the variants examined are quite low, and might therefore limit the power of future genetic association studies of tobacco dependence based on these variants.