Humans exhibit substantial inter-individual differences in TNF-alpha production upon endotoxin stimulation. To determine to what extent the lipopolysaccharide-induced TNF-alpha production capacity in vivo and ex vivo is determined by polymorphisms in toll-like receptor-4 (TLR4), the TNF-alpha promoter region and Nod2, we screened for two TLR4 polymorphisms, a Nod2 polymorphism and the TNF-alpha promoter polymorphisms. We measured the perioperative endotoxemia and TNF-alpha production and the TNF-alpha production capacity of each patient in a whole-blood stimulation assay using blood drawn before anesthesia, using various LPS concentrations, in patients undergoing elective cardiac surgery. This operation represents a major surgical trauma associated with ischemia-reperfusion injury and triggers an endotoxemia and profound inflammatory response. In vivo TNF-alpha production was positively correlated with the level of endotoxemia after aortic declamping; thus TNF-alpha levels were higher in patients having endotoxemia compared to patients without endotoxemia. This correlation was observed in patients with any of the genotypes studied, and did not differ between the various genotypes. In vivo TNF-alpha levels correlated best with those ex vivo after stimulation with 1000 ng/mL LPS, and the estimated maximal TNF-alpha release capacity. Subjects with the wild-type TLR4 gene had similar levels of TNF-alpha upon LPS stimulation ex vivo as compared with patients carrying Asp299Gly and/or the Thr399Ile TLR4 polymorphism. Our results indicate that polymorphisms in the TLR4 receptor, Nod2 and TNF-alpha promoter region are not strongly associated with in vivo and ex vivo TNF-alpha production capacity upon endotoxin stimulation. This suggests that in this model of natural LPS release, the variation between individuals in TNF-alpha release can only modestly be determined by genetic background (TNF-alpha promoter, Nod2 and TLR4) of the individual.