Polymorphism in the 5'-flanking region of the human insulin gene in 149 unrelated Japanese subjects [77 with non-insulin-dependent diabetes mellitus (NIDDM), 17 with insulin-dependent diabetes mellitus (IDDM), and 55 controls] was analyzed with Southern blot hybridization. We used the size of the hybridized fragments to classify the locus into three groups according to Bell's method (a short, class 1 allele averaging 570 base pairs; an intermediate-size, class 2 allele averaging 1320 base pairs, and a long, class 3 allele averaging 2470 base pairs in size). The allelic frequency of classes 2 and 3 in 298 alleles was 5.0% and in the 146 alleles of NIDDM, 7.8%. The value is lower than in Caucasians, American Blacks, and Pima Indians, and the results suggest that the 5'-flanking insertion is not a genetic marker in most NIDDM patients who are Japanese. However, the frequency of the 5'-flanking insertion in those who were not obese and had a family history of diabetes was higher than that of other NIDDM patients (P = .013), and the frequency in NIDDM patients with onset of diabetes at age less than or equal to 39 yr was lower than those whose onset was at age greater than or equal to 40 yr (P = .053). As NIDDM is a heterogeneous disorder, further analysis is needed. These results suggest that we cannot completely exclude the meaning of the insertion in NIDDM. On the other hand, the frequency in IDDM was 0%, lower than in NIDDM (P = .094). Because the number of subjects studied was small, this result is speculative.