In this report we have described the purification of a human plasma phospholipid transfer protein, designated LTP-II, which displayed the following characteristics: i) facilitated both the exchange and net mass transfer of lipoprotein phospholipids; ii) did not facilitate the transfer of lipoprotein cholesteryl esters (CE) or triglycerides (TG); iii) was not recognized by antibody to the human cholesteryl ester transfer protein (LTP-I); iv) showed no amino acid sequence homology to the cholesteryl ester transfer protein (LTP-I); v) has an apparent molecular weight (Mr) of 70,000 off Sephacryl S200, and 69,000 off sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE); vi) has an apparent isoelectric point of 5.0 by chromatofocusing; and vii) when added to an incubation mixture of VLDL, HDL3, and the human plasma cholesteryl ester transfer protein (LTP-I), enhanced the observed transfer of cholesteryl esters from HDL3 to VLDL, even though LTP-II has no intrinsic cholesteryl ester transfer activity of its own. These results show that this phospholipid transfer protein is unique from the human plasma cholesteryl ester transfer protein, and may play an important role in human lipoprotein lipid metabolism.