Phenotyping of cytochrome P450 2A6 (CYP2A6) was determined by assessing urinary caffeine metabolites in a Japanese population with a high frequency of CYP2A6 whole-gene deletion (CYP2A6*4). The levels of 1,7-dimethyluric acid (17U), 1-methylxanthine (1X), and 1,7-dimethylxanthine (17X) were measured in non-smokers whose CYP2A6 and NAT2 genotypes had been determined. Low 17U/1X ratios were observed in accumulated overnight urine samples of subjects genotyped as CYP2A6*4/*4 after caffeine treatment. The individual 17U/1X ratios in spot urine samples were almost constant before and 2-8 h after caffeine treatment, with or without prior abstention from dietary caffeine. The average 17U/1X ratios obtained from subjects with CYP2A6 *4/ *4 or CYP2A6 *1/ *4 genotypes were significantly lower than those from subjects with wild-type CYP2A6 *1/ *1 under dietary caffeine consumption. The present results suggest that impaired CYP2A6 function associated with CYP2A6 *4/ *4 could be determined using the 17U/1X ratios in spot urine samples under normal dietary caffeine consumption in Japanese non-smokers, without the need for additional caffeine administration or prior abstention from caffeine.