IL-23 is a novel cytokine that promotes the proliferation of naive and memory T cells and stimulates their IFN-gamma production. Besides functional similarities, IL-23 bears structural resemblance to IL-12. Biologically active IL-23 is a heterodimer whose p40 subunit is identical to IL-12p40 while its p19 subunit is distantly related to IL-12p35. In the present study we demonstrate that human monocyte-derived macrophages are able to produce IL-23 in response to virus infection. Sendai virus stimulates the expression of p19 and p40 mRNAs in macrophages. Furthermore, it enhances p35 mRNA expression and the production of IL-12. Influenza A virus, in contrast, fails to stimulate IL-12 or IL-23 expression in macrophages. IL-12 and IL-23 contribute to the IFN-gamma-inducing activity that cell culture supernatant from Sendai virus-infected macrophages show in NK-92 cells. The induction of IFN-gamma production occurs in concert with IFN-alphabeta and IL-18, which are also secreted from the virus-infected cells. The IFN-gamma-inducing activity is inhibited by IL-4, which down-regulates the transcription of p19 and p40 genes and the secretion of IFN-alphabeta, IL-12, and IL-18. IFN-gamma, in contrast, up-regulates the p19 and p40 mRNA expression in Sendai virus infection. Thus, IL-4 and IFN-gamma serve as opposing factors in the regulation of IFN-gamma-inducing cytokines, including IL-23, in macrophages.