Hyperinsulinemia, which is considered a hallmark of insulin resistance, precedes the development of non-insulin-dependent diabetes mellitus (NIDDM). Results of family and twin studies have shown that heredity influences insulin resistance and insulin levels. In Caucasian families ascertained through two or more NIDDM siblings, it has been reported that single genes with large effects, i.e., major genes, influence both fasting and 1-h postchallenge insulin levels. To determine whether a major gene affects 2-h postchallenge insulin levels in Mexican-Americans, we conducted segregation analyses using data collected on 527 pedigreed individuals from 27 families in San Antonio, TX. Probands for the families were randomly ascertained and all first-, second-, and third-degree relatives aged 16 years and older were invited to participate. Subjects received a 2-h oral glucose tolerance test, and diabetes was diagnosed according to World Health Organization criteria. We found that an autosomal dominant major gene best described the inheritance of 2-h insulin levels (ln-transformed) in these 27 families. Of the individuals in the population, 17% were homozygous for the 2-h low-insulin allele (back-transformed mean = 125 pmol/l) and 83% were heterozygous or homozygous for the 2-h high-insulin allele (back-transformed mean = 406 pmol/l). This major gene accounted for 31% of the variance in ln(2-h insulin levels) in this population. Using quantitative trait linkage analyses, we excluded tight linkage between this gene affecting 2-h insulin levels and three candidate loci for insulin levels: the insulin receptor gene, the low-density lipoprotein receptor gene, and the glucokinase gene. In addition, it is likely that this major gene is not linked to the fatty acid-binding protein gene, a gene that has previously been associated with insulin resistance through sib-pair analysis.