Plasma phospholipid binding to cell-derived cholesterol is important in reverse cholesterol transport, a key step in the regression of atherosclerosis. However, the mechanism by which phospholipids are transferred from cells to plasma remains unclear. [3H]Choline-labeled phospholipid efflux from fibroblasts has been studied using plasma and its components as acceptors. The kinetics were resolved into a fast component (k1 = 0.119 +/- 0.23 min-1) that corresponded to high-affinity binding of high-density lipoproteins (HDL) to the cell surface and a slow component (k2 = 0.0047 +/- 0.0009 min-1) due to protein-mediated desorption (n = 3). Altering the donor charge with heparinase or the acceptor charge by acetylation abolished the fast component, while the slow phase was unchanged. Only HDL displayed biexponential kinetics, comparable to whole plasma. Half-lives for low-density lipoprotein and very-low-density lipoprotein were t1/2 = 278 +/- 22 min and t1/2 = 1003 +/- 147 min, respectively. In the absence of transfer factor, HDL alone significantly reduced phospholipid efflux (t1/2 = 663 min). Phospholipid transfer protein restored biexponential kinetics. We conclude that cell membranes are a potentially important source of plasma phospholipids and that protein-mediated transfer to HDL is the major route for cell-to-plasma transfer. This step represents a locus for anti-atherosclerotic intervention.