Interleukin-18 (IL-18) enhances interferon gamma (IFNgamma) production and natural killer (NK) cell activity, and elicits protective antitumor effects in vivo. IL-18 and IL-12 synergistically augment IFNgamma production reportedly because IL-12 enhances IL-18 receptor (IL-18R) expression. We now show that IL-18 also synergizes with IL-10 to augment murine splenic NK activity against Yac-1 cells in a standard 4-h chromium-release assay, but IFNgamma production is only slightly enhanced. This pattern of NK activity was also observed with severe combined immunodeficient (SCID) mouse spleen cells indicating that the cytokines were not acting on T or B cells. The cytokines had no priming activity on the spleen cells and, when cells were left unstimulated for 24 h in culture, little NK activity was induced when IL-18 was added for the next 24 h. The reverse transcriptase/polymerase chain reaction revealed that IL-18 receptor (IL-18R) mRNA was expressed early during in vitro spleen cell culture but none was expressed after culture for 24 h regardless of the stimulus. Binding of 125I-labeled IL-18 revealed that exposure to IL-10 only slightly increased IL-18R expression. Expression of perforin mRNA was constitutive and was unaffected by the cytokines; however, Fas ligand (FasL) mRNA expression was strong in cultures with IL-18 alone or combined with IL-10. When Fas-expressing cells and their parental cells were used as targets, weak Fas-mediated cytolytic activity was observed after exposure to IL-18, and this was further enhanced by combination with IL-10. Finally, the augmentation of NK activity was abrogated by the inhibitor concanamycin A, indicating that the enhanced NK activity is perforin-dependent.