The 5' flanking polymorphism (5'FP) is a minisatellite, variable number of tandem repeat (VNTR) locus adjacent to the 5' end of the insulin gene (INS). Alleles of the 5'FP are highly variable in length but fall into three discrete size classes. The shortest, or class 1, alleles are associated with insulin-dependent diabetes mellitus (IDDM). Here we present a polymerase chain reaction (PCR)-based technique for subtyping 5'FP class 1 alleles by determining their exact lengths in number of repeat units (RUs). The technique resolves small length differences not detectable by Southern blot and produces a frequency distribution of class 1 allele lengths, which serve as subtypes of the crude class 1 category. We have applied the technique to 132 Caucasian families with IDDM offspring and have found that the lengths of 5'FP class 1 alleles form a quasi-continuous distribution with three distinct modes. We also found precise correlation between class 1 allele length and the allele present on the same chromosome at HUMTH01, a second VNTR locus in the INS region. Specifically, each of the four common alleles of HUMTH01 exhibited near-total association with a narrow size range belonging to one of the three components of the class 1 distribution. We discuss these results in relation to the population history of the 5'FP and INS region haplotypes and in relation to IDDM susceptibility in the INS region.