Hexokinase II (HKII) is the predominant hexokinase isozyme expressed in insulin-responsive tissues. Since defects involving glucose transport and/or its phosphorylation to glucose-6-phosphate are present in muscle of insulin-resistant humans, HKII should be viewed as a candidate gene for inherited insulin resistance and susceptibility to non-insulin-dependent diabetes mellitus (NIDDM). To investigate the prevalence of potential mutations in the gene encoding HKII, we used the polymerase chain reaction (PCR) to amplify each of the 18 exons of the HKII gene from genomic DNA derived from 59 subjects: 25 insulin-resistant probands with clinical features of the type A syndrome and 34 NIDDM subjects enrolled in the United Kingdom Prospective Study of Therapies of NIDDM (UKPDS) who represented the highest percentile of fasting hyperinsulinemia in the UKPDS population of 5,098 subjects. PCR products corresponding to individual HKII exons derived from each subject were screened for the presence of nucleotide variation using a sensitive nonradioactive single-strand conformation polymorphism (SSCP) protocol. Variant SSCP patterns indicative of genetic variation were detected only in PCR amplimers containing exons 4-7, 10, 15, and 17. Direct sequencing of amplified DNA from individuals affected with variant SSCP patterns revealed the presence of the following silent polymorphisms: Asp251 (GAT/C) in exon 7 and Asn692 (AAT/C) in exon 15. SSCP variants detected in PCR products containing exons 5, 10, and 17 were due to single base substitutions in flanking intronic sequences. A polymorphic GGA repeat was identified within intron 5.(ABSTRACT TRUNCATED AT 250 WORDS)