Thioglycolate-elicited mouse peritoneal macrophages were Incubated for 24 hours in serum-free Dulbecco- Vogt medium containing 0.5% fatty acid-poor bovine serum albumin. This conditioned medium, designated MP medium, was used for experiments with bovine aortic smooth muscle cells (SMCs) or human skin flbroblasts (HSFs). Dulbecco-Vogt medium of the same albumin content but without macrophages served as a control medium. In SMCs labeled from plating with [3H] cholesterol and incubated with hypercholesterolemic rabbit β-very-low-density lipoprotein O-VLDL) in Dulbecco-Vogt medium for 24 hours, there was an increase in cellular [3H] cholesteryl ester (CE) content compared with cells incubated without lipoprotein. When MP medium was used for the incubation of SMCs with β-VLDL, cellular [3H]cholesteryl ester content increased threefold compared with cells incubated with Dulbecco-Vogt medium. A smaller increase in cholesterol esterification in the presence of MP medium was also encountered with low-density lipoprotein (LDL). The MP medium-induced increase in [3H]cholesterol esterification was not evident up to 6 hours of incubation. Similar results were also obtained with HSFs. The increase in [3H] cholesterol esterification with MP medium in the presence of β-VLDL was also elicited in cells obtained from LDL receptor-negative donors with familial hypercholesterolemia (FH-HSF), even though in these cells significantly less [3H]cholesteryl ester was formed in the presence of 0-VLDL. MP medium contains numerous agents that could be responsible for the increase in cellular [3H] cholesteryl ester induced by lipoproteins. The first considered was lipoprotein lipase, but lack of inhibition of the MP medium effect by antiserum to lipoprotein lipase did not support this possibility. Recombinant apoprotein E, when added to β-VLDL and Dulbecco-Vogt medium, failed to mimic the MP effect, which argued against apoprotein E's being the "active" agent Since the increase in cellular [3H]cholesteryl ester in the presence of 0-VLDL and MP medium was more prominent in normal HSFs and SMCs than in FH-HSFs, it appears that although the LDL receptor may play a role, it is not absolutely necessary. The exact mode by which MP medium enhances cellular [3H] cholesteryl ester formation in the presence of 0-VLDL has not been elucidated, but it is important to conclude that this may occur through multiple pathways involving both the uptake of the whole particle and preferential uptake of the lipid moiety.