Revised Manuscript Received September 8, 1987 abstract: Cultures of human skin fibroblasts were labeled to high cholesterol specific activity with [3H] cholesterol and incubated briefly (1-3 min) with normal human plasma. The plasma was fractionated by two-dimensional agarose-polyacrylamide gel electrophoresis and the early appearance of cholesterol label among plasma lipoproteins determined. A major part of the label at 1-min incubation was in a pre-/?-migrating apo AI lipoprotein fraction with a molecular weight of ca. 70 000. Label was enriched about 30-fold in this fraction relative to its content of apo AI (1-2% of total apo AI). The proportion of label in this lipoprotein was strongly correlated with its concentration in plasma. Further incubation (2 min) in the presence of unlabeled cells demonstrated transfer of label from this fraction to a higher molecular weight pre-/3 apo AI species, to low-density lipoprotein, and to the a-migrating apo AI that made up the bulk (96%) of total apo AI in plasma. The data suggest that a significant part of cell-derived cholesterol is transferred specifically to a pre-/3-migrating lipoprotein AI species as part of a cholesterol transport transfer sequence in plasma.(Cholesterol fromperipheral cell membranes is available for transport into interstitial fluids and plasma. It is transferred over a period of time into all the major plasma lipoprotein fractions. However, the mechanism involved in the incorporation of cell membrane cholesterol into plasma lipoproteins is known only in outline. High-density lipoprotein (HDL), and in particular its major protein, apolipoprotein AI (apo AI), appears to play a particularly important role as an ac-ceptor of cell-derived cholesterol, as an acceptor of diffu-sion-limited efflux (Phillips et al., 1980) or, more directly, as a ligand for a receptor-mediated interaction with the cell surface (Oram et al., 1983; Graham & Oram, 1987). Other recent data implicate a minor subfraction of HDL containing