The rate of the non-directional transfer of cholesteryl ester and triglyceride by human cholesteryl ester transfer protein (CETP) was measured between human plasma lipoproteins by monitoring fluorescence spectrum of pyrene-labeled lipid. The transfer rates between high density lipoproteins (HDLs) and between low density lipoproteins (LDLs) were both directly proportional to the substrate lipid concentration within the physiological range of the lipoprotein concentration. Higher preference of cholesteryl ester transfer to triglyceride was demonstrated with HDL than LDL. Although the highly selective binding of CETP to HDL was observed in the electrophoretic analysis, the transfer rate was only moderately higher with HDL for cholesteryl ester and not so at all for triglyceride. In addition, the rate of cholesteryl ester transfer between LDLs was uninfluenced by the presence of a small amount of HDL that is just sufficient to absorb all the CETP in the reaction mixture. The results indicated the preferential transfer of cholesteryl ester over triglyceride by CETP in the interaction with HDL in non-directional lipid transfer reaction among lipoproteins. However, the apparent binding of CETP to HDL does not seem to play an essential role in this type of lipid transfer by CETP.