The functional properties of infiltrating macrophages (Mφ) must be tightly regulated to facilitate appropriate responses to complex conditions in an inflammatory focus. This study was designed to ascertain whether uncommitted Mφ that have been exposed to combinations of cytokines with opposing functions develop properties dictated by one cytokine or by cytokine mixtures. Uncommitted rat bone marrow-derived Mφ (BMDMs) were incubated with IFN-γ, TNF-α, TGF-β, IL-4, IL-6, and IL-10 alone or sequentially in combinations. After 48 h, function was assessed by nitric oxide (NO) generation, uptake of apoptotic neutrophils, and β-glucuronidase expression. IFN-γ followed 4 h later by TNF-induced NO generation. The pretreatment of BMDMs before IFN-γ priming with TNF, TGF-β, and IL-4 suppressed NO generation by 87%, 92%, and 85%, respectively; IL-10 had no effect. The same cytokines administered at 4 h after IFN priming had no effect on NO generation. The uptake of apoptotic polymorphonuclear leukocytes was augmented by TNF (40% vs 29% controls; p< 0.05) and decreased by IFN-γ, IL-10, and IL-4. The TNF response was unaffected by subsequent treatment with IFN-γ, IL-4, or IL-10. Similarly, the decreased polymorphonuclear leukocyte uptake induced by IFN-γ, IL-4, or IL-10 was unaffected by the subsequent addition of TNF. β-glucuronidase expression was increased by TGF-β and decreased by IFN-γ. These responses were not modified by cytokines with the opposing function. Thus, the functional response of BMDMs to complex mixtures of cytokines was determined by the first cytokine to which they were exposed. Once activated, BMDMs become unresponsive to alternative activating signals, a finding which has obvious implications for Mφ function in vivo.