Thioglycolate-elicited peritoneal macrophages from normal C57B1/6J mice were examined in vitro for bacterial lipopolysaccharide (LPS)-stimulated interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF) production. Macrophages from mice administered a single oral dose of levamisole (3 mg/kg) 1 to 4 days prior to macrophage harvest demonstrated a twofold enhancement of IL-1 production compared to vehicle-treated mice. In contrast, IL-6 production and TNF production by the same macrophages were inhibited up to 36 and 62%, respectively, compared to production by macrophages harvested from vehicle-treated mice. Similar results were observed when IL-1 production and TNF production were followed in peritoneal exidate cells directly stimulated with levamisole in vitro. The ex vivo LPS-stimulated IL-1 production was enhanced 4 days after macrophage elicitation, whereas TNF and IL-6 production returned to baseline by 72 h after macrophage recruitment and augmentation. No evidence could be found for the presence of inhibitors of TNF or IL-6. The specificity of the IL-1, IL-6, and TNF bioactivities was demonstrated by neutralization with specific antisera. Immunoprecipitation studies of supernatants from biosynthetically labeled macrophages also revealed augmented IL-1 production and decreased IL-6 and TNF, indicating that levamisole may have affected cytokine production at the translational level. Kinetics studies revealed that ex vivo release of IL-6 and TNF by macrophages from levamisole-dosed mice was delayed compared to production of these cytokines by macrophages harvested from mice given vehicle only. The results may explain, in part, the reported ability of levamisole to ameliorate cases of rheumatoid arthritis or other autoimmune and inflammatory diseases by affecting the relative levels of cytokines produced by macrophages recruited to sites of injury, which are associated with inflammation and acute-phase protein synthesis.