Cholesteryl ester-laden macrophages are the hallmark of the fatty streaks that precede arteriosclerotic plaques in humans and experimental animals. This article studies several aspects of cytoplasmic cholesteryl ester metabolism in cultured human monocyte-derived macrophages. Adenosine 3',5'-cyclic monophosphate (cAMP) consistently inhibited cholesteryl ester mobilization from cells that had been loaded with cholesteryl esters by preincubation with acetylated low-density lipoprotein. This effect was observed in both the absence and presence of extracellular cholesterol acceptors as well as with acyl coenzyme A: cholesterol acyltransferase inhibitors. In contrast, dibutyryl cAMP activated cholesteryl ester hydrolysis in J774 macrophages. Since hormone-sensitive lipase is thought to be responsible for the neutral cholesteryl ester hydrolytic activity in several cell types, we looked for the presence of its mRNA in our macrophages by means of reverse transcription coupled to the polymerase chain reaction technique. Hormone-sensitive lipase mRNA was detected in J774 macrophages but not in human monocytes or in human monocyte-derived macrophages. These results demonstrated great differences in cholesteryl ester metabolism between macrophages of different origin. While hormone-sensitive lipase may be responsible for neutral cholesteryl ester hydrolytic activity in J774 macrophages, in human monocyte-derived macrophages it is not; thus, a different and as yet unidentified enzyme must be present.