A widely applied method to study the activation of the innate immune system is in vitro stimulation of whole blood using lipopolysaccharide (LPS). However, it is unclear if in vitro cytokine production relates to in vivo cytokine levels elicited during experimental endotoxemia or sepsis. To determine the correlation between in vitro cytokine production and the in vivo inflammatory response, blood was obtained from 15 healthy volunteers for in vitro incubation with Escherichia coli LPS, immediately followed by experimental E. coli endotoxemia. Correlations of in vitro and peak in vivo cytokine concentrations were determined using Pearson correlation coefficient. In stimulated whole blood, tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6, IL-10 and interferon (IFN)-γ were induced to 279 ± 53, 392 ± 64, 5312 ± 624, 83 ± 20 and 343 ± 85 pg/ml, respectively, whereas in vivo cytokine induction led to cytokine levels of 603 ± 123, 11 ± 1, 4999 ± 1228, 167 ± 25 and 194 ± 40 pg/ml, respectively. Correlation coefficients between the in vitro and in vivo cytokine concentrations were for TNF-α, IL-1β, IL-6, IL-10 and IFN-γ -0.10 (P = 0.7), 0.09 (P = 0.8), 0.36 (P = 0.2), 0.19 (P = 0.5) and 0.40 (P = 0.1), respectively. Comparison between in vitro and in vivo stimulation with LPS shows no correlation between the amount of cytokines produced. In vitro cytokine production, therefore, does not predict the in vivo inflammatory response.