Interleukin-1beta (IL-1beta) activates inflammatory mediator cascades and has been implicated in the pathogenesis of several diseases. Single nucleotide polymorphisms (SNPs) of the IL1B promoter have been associated with various inflammatory diseases. We recently reported that IL1B gene transcription was influenced by four promoter SNPs, and that individual SNP function in vitro was governed by haplotype context. In the present study we tested the in vivo relevance of this observation by comparing IL1B promoter haplotype-pairs with IL-1beta protein levels in 900 gingival tissue fluid samples. Three SNPs (-511, -1464, -3737) defined four IL1B promoter haplotypes that occurred in the study population and could be assigned unambiguously to each chromosome. The four haplotypes defined ten haplotype-pairs of which four pairs, representing 57% of the population, were associated with 28-52% higher IL-1beta protein levels in vivo. Two of these pairs, characterized by homozygosity for the common allele at -3737, were also associated with raised serum levels of C-reactive protein (p = 0.02). We validated these findings in stimulated peripheral blood mononuclear cells (PBMCs) from a separate population (N = 70). PBMCs with IL1B haplotype-pairs associated with higher in vivo levels of IL-1beta produced 86-287% more IL-1beta in vitro than the reference group. We believe that this is the first demonstration of a relationship between in vivo levels of an inflammatory mediator and gene promoter haplotypes on both chromosomes. These findings may apply to other inducible genes and could provide a logical framework for exploring disease risk related to genetic variability in pathogenic mediators.