Mice deficient in both macrophage-colony-stimulating factor (M-CSF, op) and apolipoprotein E (apoE) have elevated cholesterol levels but are protected from atherosclerosis. To assess the contribution of macrophage (Mphi) phenotypic heterogeneity and scavenger receptor (SR-A) expression to this seeming paradox, we characterized the Mphi phenotype by immunohistochemistry in these animals. Lesion size was determined in animals fed a chow or Western-type diet, and lipoprotein clearance studies were performed in vivo. Op0/E0 mice have fourfold smaller aortic root lesions than op2/E0 animals despite 2.5-fold higher total plasma cholesterol levels. Mphis in atherosclerotic lesions of op2/E0 mice constitute a predominantly recruited and M-CSF-dependent population. In addition, Mphis in different locations in plaques show phenotypic heterogeneity. SR-A expression in op0/E0 mice is reduced in proportion to the decrease in Mphi numbers, and M-CSF is thus not an essential requirement for SR-A expression in vivo. M-CSF-deficient mice degrade injected AcLDL , showing an adequate level of SR-A activity present in vivo. In contrast, beta-VLDL clearance in op0/E0 mice is decreased, implicating monocytes/Mphis in its catabolism. There is prominent lipid accumulation in op2/E0 Kupffer cells and hepatocytes but not in M-CSF-independent Kupffer Mphis from op0/E0 mice. SR-A, while abundantly expressed on both Kupffer cells and sinusoidal endothelial cells in op2/E0 mice, remains mainly on sinusoidal endothelial cells in op0/E0 mice. This may explain preservation of SR-A activity in these animals. Our findings clearly illustrate the importance of both M-CSF and M-CSF-dependent monocytes/Mphis in maintaining cholesterol homeostasis and in atherogenesis.