Cardiopulmonary bypass (CPB) is associated with immune paresis, which predisposes to the development of postoperative sepsis. The aims of this study were to characterize the ex vivo cytokine responses to bacterial cell wall components in whole blood from patients undergoing CPB and to determine whether altered leukocyte expression of Toll-like receptors (TLRs) is involved in immune paresis after CPB. We recruited 6 patients undergoing routine cardiac surgery with CPB. Preoperatively, at the end of CPB and 20 h later, blood was obtained, anticoagulated, and leukocyte surface expression of CD14, TLR2, and TLR4 was quantified by flow cytometry. In addition, blood was incubated at 37 degrees C in the presence of peptidoglycan (PepG) and/or lipopolysaccharide (LPS), and plasma cytokines were measured by enzyme immunoassay. At the end of CPB, ex vivo production of tumor necrosis factor alpha, interleukin (IL) 1beta, IL-8, and IL-10 in response to PepG or LPS was virtually abolished (P < 0.05). The following day, there was recovery of all cytokine responses to PepG. Tumor necrosis factor alpha and IL-1beta responses to LPS partially recovered, whereas IL-8 and IL-10 responses recovered. At the end of CPB, there was more than 50% reduction in neutrophil TLR2 and TLR4 expression (P < 0.05), with recovery to baseline the following day. There was a 29% reduction in monocyte TLR4 expression at the end of CPB (P < 0.05) and more than 120% increase in monocyte TLR2 and 4 expression the following day (P < 0.05). In conclusion, reduced ex vivo production of cytokines cannot be fully accounted for by downregulation of TLR expression, although receptor upregulation may contribute to the later recovery of responsiveness.