We have studied cholesteryl ester accumulation in human monocyte-derived macrophages, which together with smooth muscle cells, represent the major cell types that accumulate cholesterol in atherosclerotic lesions. Monocyte-derived macrophages were incubated with either acetylated low density lipoprotein (AcLDL) or non-lipoprotein cholesterol and the question as to whether all of the cells, or specific cell subpopulations could accumulate cholesteryl ester was examined. We stained cholesteryl ester in monocyte-macrophages with the fluorescent probe filipin. Cholesteryl ester accumulated as lipid droplets that were widely dispersed in the cell cytoplasm. Interestingly, no more than 65% of monocyte-macrophages accumulated cholesteryl ester during the 1st day of incubation with non-lipoprotein cholesterol. By 2 days of incubation, greater than 90% of cells displayed cholesteryl ester deposition. The cholesteryl ester which accumulated during the 2nd day of incubation was derived from unesterified cholesterol that had accumulated during the 1st day of incubation. This finding was substantiated by the following: (1) chemical measurements showed that the total cholesterol content of monocyte-macrophages did not increase further after the 1st day of incubation, and (2) all monocyte-macrophages had accumulated fluorescent tagged cholesterol during the 1st day of incubation. In contrast to the results obtained with non-lipoprotein cholesterol, more than 90% of monocyte-macrophages incubated with AcLDL for 1 day accumulated cholesteryl ester in two experiments. However, less than 62% of monocyte-macrophages accumulated cholesteryl ester in two other experiments, thereby resembling results obtained with non-lipoprotein cholesterol. Again, the lack of cholesteryl ester accumulation with AcLDL was not due to a lack of uptake of AcLDL, as greater than 90% of monocyte-macrophages accumulated fluorescent tagged AcLDL. The observed heterogeneity in cholesterol esterification among human monocyte-macrophages suggests that functional subpopulations of these cells may exist with respect to cholesterol processing. However, heterogeneity in cholesteryl ester accumulation did not seem to correlate with expression of HLA-DR antigen, a marker of immunological activation of macrophages. Other sources of heterogeneity most likely result from inter-cellular variation at one or more levels of regulation of the cholesterol trafficking and esterification process.