Aim To study the mechanism of cholesterol efflux from rat peritoneal macrophages mediated by high density lipoprotein-3(HDL 3). Methods Modification of HDL 3 by N-acetylimidazole blocked interaction between HDL and its cellular receptor. FITC labeled HDL 3 were used to observed the cellular metabolic process of HDL 3. Results BSA (control group) mediated 2.9% cellular cholesterol efflux from the cells. In HDL 3 group and N-acetylimidazole-HDL 3 group,they were 40.7% and 8.7% respectively. After incubation of macrophages with FITC-HDL 3 at 37℃for 3 h, the cell-endocytic fluorescence strength (FS) was 65.0% of the cell-associated FS. When the cells were further incubated with blank media at 37℃for 2 h, 78.4% of the cell-endocytic FS was released into the media. Both the cell-endocytic FS and the cell-released FS were mainly in trichloroacetic acid precepitable form. Conclusions The cellular cholesterol efflux from macrophages mediated by HDL 3 was HDL receptor-dependent. The possible mechanism could be that macrophages internalized HDL 3 by HDL receptor. HDL 3 picked up cellular cholesterol and was resecreted out of cells by a retroendocytic pathway without taking a celluar lysosomal phathway.