Hypertriglyceridemia is a common metabolic disorder with a major inherited component. In some individuals the condition is suspected to occur as a result of overproduction of apolipoprotein (apo)CIII, a major constituent of triglyceride-rich lipoproteins. Population studies have established an association with the apoCIII gene but the identify of the causal mutation remains unknown. In the present study we have examined a series of six 5' polymorphic nucleotides (G-935 to A, C-641 to A, G-630 to A, deletion of T-625, C-482 to T, and T-455 to C) that lie within the promoter region of the apoCIII gene for evidence of possible involvement in disease susceptibility. The polymorphic nucleotides at positions -455 and -482 reside within a negative insulin-response element. We show, in a community-based sample of 503 school children, that a DNA polymorphism (S2 allele) within the 3'-noncoding region of the apoCIII gene was associated with elevated apoCIII and triglyceride levels, but that the polymorphic nucleotides of the promoter were not. In addition, no obvious effect of any extended apoCIII promoter haplotype on plasma apoCIII or triglyceride levels, over and above that conferred by the presence of the S2 polymorphic nucleotide, was detected. These results demonstrate that none of the 5' apoCIII polymorphisms can account for the association of the apoCIII gene locus with hypertriglyceridemia and, moreover, owing to linkage disequilibrium, raise the possibility that the region conferring susceptibility maps downstream, rather than upstream, of the apoCIII gene promoter sequences.