A compound imperfect dinucleotide repeat element, [CA]4TTTGT[CT]7[CA]9AA[CA]4CCACATA[CA]3, was found approximately 10 kb 3' to the human glucokinase gene (GCK) from analysis of contiguous genomic DNA obtained from a bacteriophage lambda chromosome walk. Direct human genomic sequencing revealed the source of polymorphism to be variable numbers of CT and CA repeats. Altogether six alleles that range in length from +10 to -15 nucleotides compared to the most common (Z) allele have been identified. Alleles Z, Z + 2, and Z + 4 were present in American Blacks, Pima Indians, and Caucasians, with somewhat varied frequencies among the groups. Two alleles, Z + 10 and Z - 15, appear to be unique to American Blacks, while a Z + 6 allele was observed only in the Caucasian population studied. Observed heterozygosity of the polymorphism in the CEPH reference pedigree collection is 44% and the PIC 0.44. The polymorphism is assayed by PCR amplification and resolution of 32P-end-labeled products (ranging in length from 180 to 205 bp) on denaturing polyacrylamide sequencing gels. Using the PCR assay, the human glucokinase gene was physically localized to chromosome 7 in a panel of rodent/human somatic cell lines. Genetic analysis in CEPH pedigrees placed the dinucleotide repeat element, and thereby the human glucokinase gene, on chromosome 7p between TCRG and a RFLP locus D7S57. The glucokinase dinucleotide repeat genetic marker can now be used to assess the role of the glucokinase gene in diabetes by population association studies. In addition, this repeat marker and others flanking it on chromosome 7 can be used in linkage studies with families segregating the disorder.