While some studies clearly demonstrated transfer of cytokine-inducing substances (CIS) through dialysis membranes, other authors were unable to reproduce such a transfer. This inconsistency may have been caused by marked differences in experimental design. We performed a systematic evaluation of cytokine induction in whole blood and from separated peripheral blood mononuclear cells (PBMC) using either purified lipopolysaccharide (LPS) or culture supernatants from various bacterial strains. An in vitro hemodialysis circuit with whole blood in the blood compartment was employed; the dialysate was contaminated with sterile filtrates from Pseudomonas aeruginosa cultures. Addition of plasma samples from the blood side to PBMC readily induced interleukin (IL)-1β and IL-6 after contamination of the dialysate, while the same samples failed to induce cytokine production in whole blood. In experiments using direct incubation, purified P. aeruginosa LPS induced more IL-1β and IL-6 in whole blood compared to PBMC. In contrast, bacterial filtrates from Escherichia coli, Enterobacter cloacae and especially P. aeruginosa induced significantly less cytokines in whole blood compared to PBMC. Addition of erythrocytes but not granulocytes decreased cytokine induction by bacterial filtrates as well as by LPS, probably by adsorption of these substances. The addition of 30% plasma increased cytokine induction by LPS but decreased cytokine induction by P. aeruginosas filtrates. Our results suggest that cellular and plasmatic components of whole blood interact with bacterial CIS altering their pyrogenic activity. The detection of CIS depends on the test system used; whole blood cultures are not as sensitive for the detection of CIS from bacterial filtrates as PBMC cultures.