The heterozygous Asp299Gly mutation of the toll-like receptor (TLR) 4, the key receptor for lipopolysaccharide (LPS), has been associated with attenuated inflammatory responses. When 160 healthy volunteers (9% heterozygous and 0.6% homozygous) were genotyped and their LPS-inducible cytokine release was assessed in an ex vivo whole blood test, the responses of heterozygotes did not differ significantly from those of wild-type carriers for any of the cytokines (tumor necrosis factor-alpha, interleukin [IL]-1beta, IL-6, interferon-gamma, and granulocyte colony-stimulating factor) or eicosanoids measured or for serum cytokines and C-reactive protein. Ten heterozygous subjects and 12 wild-type control subjects responded similarly to a graded series of LPS and Escherichia coli concentrations, excluding the possibility that allele-specific differences are evident only at low stimulus concentrations or in response to whole pathogens. These data demonstrate that the heterozygous Asp299Gly polymorphism does not exhibit a functional defect in cytokine release after the stimulation of blood monocytes.