The interaction between HDL and macrophages in culture was studied using HDL labeled with 125I and with [3H]cholesteryl linoleyl ether. Mouse peritoneal macrophages and the macrophage-like cell lines J-774 and CT2, of mouse origin, took up and metabolized rat HDL and human HDL3. In all 3 cell types using both rat and human HDL, the uptake of the cholesteryl ester moiety as measured with the nondegradable cholesteryl ether analog, was 2-5-fold higher when compared to the protein moiety. Modulation of the cholesterol content of the cultured macrophages affected the uptake of both protein and lipid moieties of HDL to the same extent. When the macrophages had interacted with the labeled HDL for 5 h and were post-incubated for 20 h, the amount of [125I]HDL which reappeared in the post incubation medium was twice that of [3H]cholesteryl linoleyl ether-HDL. The site from which the HDL may have returned to the culture medium was tentatively localized to the trypsin-releasable, cell surface-related compartment. The present results indicate that interaction between macrophages and HDL may result in some loss of cholesteryl ester and possibly render the particle more receptive for cellular cholesterol removal.