Colony-stimulating factor 1 (CSF-1) can act on mature macrophages to modulate their production of inflammatory cytokines. A cDNA encoding the interleukin-1 receptor antagonist (IL-1Ra) was cloned by subtractive hybridization from a CSF-1-stimulated murine macrophage cell line, sequenced, and expressed in mammalian and bacterial cells. Mouse IL-1Ra is a 22-Kd glycoprotein that is 76% identical to its human counterpart, shows considerably less similarity to IL-1 alpha and IL-1 beta, and competes with IL-1 alpha for binding to the type I IL-1 receptor normally expressed on T cells and fibroblasts. CSF-1 treatment of mouse bone marrow-derived macrophages led to a rapid and sustained increase in IL-1Ra mRNA during the G1 phase of the cell cycle as well as to increases in mRNAs encoding IL-1 alpha and IL-1 beta. Cycloheximide inhibited CSF-1-induced IL-1 alpha mRNA synthesis, but augmented IL-1 beta mRNA production and did not affect induction of IL-1Ra mRNA. No IL-1Ra mRNA was observed in CSF-1-stimulated mouse fibroblasts engineered to express CSF-1 receptors, demonstrating that its regulation depends on cell context and can be dissociated from the proliferative response. In agreement, bacterial lipopolysaccharide, a nonmitogenic activator, also induced IL-1Ra and IL-1 mRNAs in macrophages. Unlike IL-1 alpha and beta, IL-1Ra contains a signal peptide. The kinetics of its induction and its ability to gain access to the secretory compartment imply that IL-1Ra may be secreted more efficiently than IL-1, and suggest that macrophages both positively and negatively regulate the IL-1 response.