Cystatins are natural tight-binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon-γ-activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077–28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon-γ and then stimulated with interferon-γ plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon-γ. Interferon-γ-activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon-γ plus chicken cystatin produced increased amounts of both tumor necrosis factor α and interleukin 10. The addition of recombinant murine tumor necrosis factor α alone or in combination with recombinant murine interleukin-10 mimicked the effect of chicken cystatin. The addition of neutralizing anti-(tumor necrosis factor α) antibodies reduced sharply NO production by chicken cystatin/interferon-γ-activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor α and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon-γ-activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.