Different macrophage preparations were compared for functional capacity in conditions of high prostaglandin E2 (PGE2) or low L-arginine concentrations. Macrophages derived in vitro from bone marrow progenitor cells (bone marrow-derived macrophages, BMDMs) using colony-stimulating factor 1 (CSF-1) as the myelopoietic stimulus displayed a greater sensitivity to PGE2-induced suppression of tumor necrosis factor α (TNF-α) secretion than did macrophages derived using granulocyte-macrophage colony-stimulating factor (GM-CSF). Neither BMDM population was inhibited by PGE2 for the direct cytolysis of L929 cells (TNF-α sensitive), and only GM-CSF-derived macrophages showed decreased killing of TNF-α-resistant K562 targets. Exogenous cAMP inhibited TNF-α secretion, but not nitrite secretion, by both BMDM populations. GM-CSF-derived macrophages accumulated less cAMP following PGE2 treatment than did CSF-1-derived macrophages. Removing L-arginine from the medium did not inhibit cytotoxicity or PGE2 secretion, but the listeriacidal activity specific to interferon-γ plus lipopolysaccharide (LPS)-activated GM-CSF-derived macrophages was blocked by removal of L-arginine. Treatment with CSF-1 or GM-CSF alone did not activate the macrophages, but GM-CSF efficiently primed both BMDM populations for augmented TNF-α secretion in response to secondary stimulation using LPS. However, GM-CSF augmented the LPS-induced production of nitrite and PGE2 by CSF-1-derived macrophages only. These results demonstrate the potential for differential macrophage function within inflammatory sites based on the hematopoietic stimulus under which the macrophage is derived and the specific conditions present in the lesion.