Two poor metabolizer individuals of debrisoquine were identified among 215 healthy Japanese by a phenotyping test. Analysis of the CYP2D6 gene from one of two poor metabolizers, who was not homozygous for the previously described CYP2D6 variant alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18), showed that this individual was heterozygous for a new allele, CYP2D6/C8 (CYP2D6*21). CYP2D6*21 had a single cytosine insertion at position 2661 in exon 5. This cytosine insertion generated a stop codon at the 17 bp downstream of this insertion site. A method to detect this allele was established with an allele specific-polymerase chain reaction. This method showed that another one of two poor metabolizers also possessed CYP2D6*21 allele heterozygously. In 318 healthy Japanese, five individuals carried this allele, heterozygously (0.81%, 5/636 chromosomes). Based on the present and our previous data, the poor metabolizer frequency in Japanese was estimated to be 0.39%, which accounted for approximately 45% of the individuals phenotyped as poor metabolizers by in-vivo tests.