Cytochrome P450 (CYP) 2A6 metabolizes a number of drugs and a variety of procarcinogens. CYP2A6 also catalyzes nicotine C-oxidation leading to cotinine formation, a major metabolic pathway of nicotine in humans. There are genetic polymorphisms in the human CYP2A6 gene and a relationship between the CYP2A6 genotype and smoking habits as well as the incidence of lung cancer has been indicated. CYP2A6*4 alleles are the whole deleted type and are completely deficient in the enzymatic activity. An unequal crossover junction is located in the 3'-flanking region in the CYP2A6*4A allele, whereas the junction is located in either intron 8 or exon 9 in the CYP2A6*4D allele. In the present study, a novel genotyping method to distinguish between two different whole deleted alleles of CYP2A6*4A and CYP2A6*4D was established. In the process, two novel alleles, CYP2A6*1F and CYP2A6*1G, were found. The CYP2A6*1F has a single nucleotide polymorphism (SNP) of C5717T in exon 8, and the CYP2A6*1G has two SNPs, C5717T in exon 8 and A5825G in intron 8. The SNP of C5717T corresponds to C1224T on the cDNA sequence and is a synonymous mutation. Since the CYP2A6*1F produces a recognition site of the restriction enzymes that is the same as CYP2A6*4D, the presence of the CYP2A6*1F allele could cause a mistyping as the CYP2A6*4D allele. According to an improved genotyping method, the allele frequencies of CYP2A6*4A, CYP2A6*4D, CYP2A6*1F, and CYP2A6*1G in 165 Caucasians were 3.0%, 0%, 1.8%, and 1.2%, respectively. The allele frequencies of CYP2A6*4A, CYP2A6*4D, CYP2A6*1F, and CYP2A6*1G in 94 African-Americans were 0%, 0.5%, 0%, and 13.3%, respectively. This is the first report of a method that can distinguish between CYP2A6*4A, CYP2A6*4D, and CYP2A6*1F which could otherwise cause a mistyping as CYP2A6*4D.