Macrophages derived in vitro from bone marrow progenitors (bone marrow–derived macrophages, BMDMs) using either macrophage colony-stimulating factor (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) as the myelopoietic stimulus display differential functional, morphological, and mRNA phenotypes. The data presented here demonstrate further that CSF-1– and GM-CSF–derived BMDMs differ in immunologic capacity. GM-CSF–derived BMDMs, when compared to CSF-1–derived BMDMs, showed greater cytolytic activity against tumor necrosis factor α (TNF-α)–resistant, but not TNF-α–sensitive, tumor targets. In contrast, CSF-1–derived BMDMs produced nitrite in response to lipopolysaccharide (LPS) alone, whereas GM-CSF–derived BMDMs required interferon γ plus LPS treatment. The two BMDM populations also showed differential sensitivities to LPS for secretion of TNF-α and nitrite, but the maximal inducible amounts of these factors and prostaglandin E₂ were similar between the BMDM populations. Lastly, GM-CSF–derived but not CSF-1–derived BMDMs showed an L-arginine–dependent listeriacidal activity. These results show that the functional heterogeneity of CSF-1– and GM-CSF–derived macrophages is limited and appears to result largely from differences in the activational signals required by each BMDM population to elicit a given function.