IL-12 is important for Th1 differentiation. Myeloid-derived antigen-presenting cells (APC) such as monocytes, macrophages (Mphi) and dendritic cells (DC) are believed to be major sources of IL-12 in vivo. We have compared IL-12 production of fresh monocytes with Mphi differentiated in vitro using macrophage colony-stimulating factor (M-CSF) or human plasma, and in vitro generated dendritic cells, since these differentiated cell types represent APC at sites of antigen challenge. Macrophages stimulated with lipopolysaccharide (LPS) or heat-killed Listeria monocytogenes in the presence or absence of IFN-gamma produced minimal IL-12 p70 by comparison with DC or monocytes, despite comparable production of TNF-alpha. M-CSF-induced Mphi produced low levels of IL-10 constitutively and high levels after stimulation with LPS, but neutralization of IL-10 did not augment Mphi IL-12 production. Exposure of Mphi to TNF-alpha, granulocyte-macrophage CSF or IFN-gamma did not substantially up-regulate IL-12. Therefore M-CSF induces a differentiated Mphi phenotype in which IL-12 production is down-regulated, perhaps irreversibly. This may be the default pathway for monocyte-Mphi development in the absence of inflammation.