Previous studies have implicated a role of bacterial DNA, containing unmethylated cytosine-phosphate-guanosine (CpG) motifs, in the initiation of systemic inflammation. This is based on the ability of CpG-DNA to act in synergy with lipopolysaccharide (LPS) to trigger tumor necrosis factor alpha (TNFalpha) production in murine monocytes and to enhance LPS toxicity in rodents. In this study we investigated the capacity of CpG-DNA to trigger and modulate cytokine responses in human leukocytes. A human blood assay, as well as isolated cultures of monocytes and neutrophils, was exposed to the synthetic oligodeoxynucleotides (ODNs) CpG ODN (2006) and GpC ODN (2006-GC), alone or in combination with peptidoglycan or LPS. Plasma or supernatants were isolated and analyzed for TNFalpha, interleukin-1 beta (IL-1beta), IL-6 and IL-8 by ELISA. In the blood, 2006 (but not 2006-GC) induced the release of TNFalpha (P < 0.05) and possibly IL-1beta and IL-6. IL-8 was induced in a CpG-independent manner. When co-administered with peptidoglycan, both ODNs enhanced the release of cytokines, but not consistently CpG dependent. When co-administered with LPS, only IL-8 values were enhanced, whereas IL-6 was suppressed at early time points. In monocyte and neutrophil cultures, CpG dependent induction of cytokine release was not observed. However, both ODNs inhibited LPS-induced IL-6. In conclusion, the capacity of CpG DNA to trigger the release of TNFalpha and to enhance LPS-induced release of this cytokine is confirmed in human whole blood, but not in adherent human monocytes. Most effects of the ODNs on cytokine release in human leukocytes were CpG independent.