Glucokinase (ATP:D-glucose-6-phosphotransferase), expressed exclusively in liver and pancreatic islet beta-cells, catalyzes the first step of glycolysis and acts as glucose sensor and metabolic signal generator in these tissues. The enzyme plays a key role in glucose homeostasis and as such is an excellent candidate for inherited defects predisposing to non-insulin-dependent diabetes mellitus (NIDDM). A compound-imperfect dinucleotide (CA)n repeat element was found approximately 10-kb 3' of the human glucokinase gene on chromosome 7p, which revealed polymorphism with alleles differing in size by 2-15 nucleotides in unrelated individuals. A polymerase chain reaction assay was developed, and genomic DNA from 275 biologically unrelated American black individuals was typed for glucokinase alleles. The differences in allelic frequencies between individuals with NIDDM and nondiabetic individuals were compared. After typing 112 diabetic and 163 nondiabetic subjects, we found five different-sized alleles, with Z defined as the most common allele, Z + 2, Z + 4, Z + 10, and Z - 15. The Z allele was more common in nondiabetic subjects than in diabetic patients (60.4 vs. 49.6%, P = 0.012). The Z + 4 allele was more common in diabetic patients than in nondiabetic subjects (20.1 vs. 12.0%, P = 0.009). After adjusting for age, sex, and body mass index, the Z + 4 allele continued to have a positive association with NIDDM (P = 0.0018), and the Z allele had a negative association with NIDDM (P = 0.0334). The Z + 4 allele, transmitted as an autosomal dominant trait, appeared to be the most significant one at this locus.(ABSTRACT TRUNCATED AT 250 WORDS)