A new 55-kDa HDL/apolipoprotein binding protein was demonstrated in plasma membrane preparations of the human cell lines and primary cultured hepatocytes, Analysis of specific binding by ligand immunoblots of HDL, apoA-I, and apoA-II to a partially purified S5-kDa PA-I plasma membrane preparation demonstrated a K-d,K-HDL = 50 nM (10 mug/ml), K-d,K-apoA-II = 20 nM (0.4 mug/ml), and K-d,K-apoA-I = 330 nM (10 mug/ml). Following preparative SDS-PAGF electrophoresis of a plasma membrane preparation isolated from human PA-I cells, fractions with apoA-II binding activity were collected, concentrated, and subjected to two-dimensional electrophoresis. Internal microprotein sequencing of the 55-kDa protein band revealed the binding protein as being heat shock protein 60 (hsp60), The hsp60 monoclonal antibody LK-1 blocked apoA-II binding to the 55-kDa HBP preparation. In summary, these results provide a potential mechanism to explain the known association between immunity developed against hsp60 and the development of atherosclerosis,