The effect of human plasma phospholipid transfer protein (PLTP) on the particle size distribution of human high density lipoprotein (HDL) was studied by incubating human HDL3 (particle diameter, 8.7 nm) together with PLTP in vitro. Incubation of HDL3 with highly purified preparations of PLTP, devoid of cholesterol ester transfer protein (CETP), induced a conversion of the homogenous population of HDL particles into two main populations of particles, one larger, particle diameter 10.9 nm, and one smaller, particle diameter 7.8 nm, than the original HDL3. These size changes were evident as analyzed by gradient gel electrophoresis and by high resolution gel filtration. The degree of the conversion was dependent on the amount of PLTP added to the incubation and on incubation time. An inhibitory monoclonal antibody (TP-1) directed against CETP had no effect on the HDL conversion. The PLTP used was purified to homogeneity from human plasma using ultracentrifugation and a combination of hydrophobic, cation-exchange, heparin-Sepharose-, anion-exchange, and gel filtration chromatographies. The monoclonal anti-CETP antibody (TP-1), which inhibits lipid transfer catalyzed by CETP, did not react with PLTP or inhibit its activity. The estimated molecular weight of PLTP is 75,000. The present study demonstrates that PLTP can act like the putative conversion factor and has the ability to convert HDL3 into populations of larger and smaller HDL particles. The mechanism(s) involved in this process and its physiological relevance remain to be established.