A specific set of immune functions is switched on in response to DNA containing unmethylated CpG dinucleotides in particular base contexts ('CpG motifs'). Plasmids, viral vectors and antisense oligodeoxynucleotides used for DNA vaccination, gene replacement or gene blockade contain immunostimulatory CpG motifs which may have independent biological activity. Although the immune stimulatory effects of CpG motifs on murine cells are well established, the evaluation of their possible effects on human cells is complicated by the higher LPS sensitivity of human leukocytes compared with those in mice. To address this issue, we analyzed CpG- and LPS-mediated immune activation of human PBMC. The biologic activity of LPS could be detected within 4 h using intracellular TNF staining of monocytes with flow cytometry at concentrations just one-twentieth (0.0014 Eu/ml) of the lower detection limit for the routinely used LAL assay (0.03 EU/ml). In contrast to the rapid LPS response, CpG DNA-stimulated TNF and IL-6 synthesis in human monocytes was not detectable until 18 h. E. coli DNA induced IL-6 synthesis in a concentration-dependent manner (30 micrograms/ml E. coli DNA; 409 pg/ml +/- 75 pg/ml, n = 7, IL-6 ELISA), but calf thymus DNA did not (< 10 pg/ml). Likewise, the CpG oligodeoxynucleotides 1760 (phosphorothioate) and 2059 (unmodified) induced IL-6 synthesis, but the corresponding control oligonucleotides 1908 and 2077 did not CpG DNA and LPS enhanced IL-6 synthesis synergistically. ICAM-1-expression of monocytes was increased 4.6-fold by E. coli DNA, 3.5-fold by 1760 and three-fold by 2059, compared with 3.6-fold by a maximal LPS stimulus and no change with non-CpG DNA. In conclusion, CpG-motifs induce TNF, IL-6 and ICAM-1 expression in human monocytes, but the kinetics of this differ from that induced by LPS, which makes it possible to distinguish immune activation by these agents. These results have important implications for the clinical development of therapeutic DNA in humans.