We have devised techniques for the isolation of human monocytes which do not require the adherence of the cells to a surface. In 15 consecutive experiments using density-gradient and counterflow centrifugations, a population of mononuclear cells that was 75 +/- 11% monocytes was obtained within 2 hours of venipuncture. These cells had never been pelleted and represented approximately three-fourths of the monocytes that had been present in the whole blood. In another 22 consecutive experiments using sedimentation in gelatin followed by counterflow and density-gradient centrifugations, a population of lymphocytes that was 99.5 +/- 0.5% pure and a population of monocytes that was 94 +/- 3% pure were obtained within 3 hours of venipuncture. When these freshly isolated cells were incubated in the lipoprotein-deficient fraction of serum (d > 1.21 g/ml) or in solvent-extracted serum, the monocytes incorporated 10-20 times more [2-(14)C]acetate into sterols than did the lymphocytes. Monocytes were seen to constitute between 6 and 46% of the mononuclear cells isolated from normal individuals by the usual density-gradient centrifugation of whole blood on Ficoll-Hypaque. We conclude that future studies of cholesterol metabolism utilizing human mononuclear cells must take into account this large variation in the percentage of monocytes and their disproportionately greater activity during short-term incubations in media that induce sterol synthesis.-Fogelman, A. M., J. Seager, M. Hokom, and P. A. Edwards. Separation of and cholesterol synthesis by human lymphocytes and monocytes.