During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes forCYP2A6*4Cand the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes forCYP2A6*4Cand theCYP2A6*1Awas carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3′-untranslated region, which was replaced with the corresponding region of theCYP2A7gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes forCYP2A6*4CandCYP2A6*1A. To investigate this further, we developed a genotyping method which could distinguish theCYP2A6*1A, CYP2A6*1BandCYP2A6*4Calleles from each other. The results clearly showed thatCYP2A6*1Bwas the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of theCYP2A6gene, simultaneously and precisely, in both Oriental and Caucasian populations.