Genetic variations contribute to differences in the inflammatory response and are markers for disease risk and outcomes. We studied three single-nucleotide polymorphisms (-597 G-->A, -572 G-->C, and -174 G-->C) in the IL-6 promoter to determine associations with ex vivo LPS-stimulated IL-6 production by leukocytes. We also measured nuclear protein binding to synthetic oligonucleotides representing the -174 polymorphic region, located in proximity to important transcription factor motifs. We determined genotypes at three sites in the IL-6 promoter by pyrosequencing of genomic DNA obtained from 49 healthy control subjects. To determine molecular haplotypes, cloned DNA fragments from heterozygous subjects were sequenced. IL-6 release by whole blood leukocytes was measured after 24 h of ex vivo LPS stimulation. We compared IL-6 concentrations between haplotypes using Kruskall-Wallis and adjusted for covariates by analysis of covariance. Electromobility gel shift assay was carried out by the incubation of nuclear proteins from cultured human mononuclear cells with oligonucleotides representing the alternate -174 alleles. The amount of nuclear protein binding was quantified by densitometry, which was compared using analysis of variance. Genotype and sequence analysis of genomic and cloned DNA characterized three haplotypes. Ex vivo IL-6 production was greatest in individuals who were homozygous for the haplotype containing guanine at -597 and -174. IL-6 production was least for individuals homozygous for the haplotype containing adenine at -597 and cytosine at -174. Nuclear protein bound more avidly to guanine-containing oligonucleotides representing the -174 position than to oligonucleotides containing cytosine at that position. The IL-6 promoter haplotype influences ex vivo IL-6 response to endotoxin. This effect may be due to a functional effect of the -174 G-->C polymorphism.