Various factors influencing the protein binding of vancomycin were examined using equilibrium dialysis method. Four per cent human serum albumin (HSA) and/or 0-08 per cent alpha-I-acid glycoprotein (AAG), dissolved in isotonic phosphate buffer, were dialyzed against isotonic phosphate buffer of pH 7-4 using Spectrapor 2 membrane. The protein binding of vancomycin to 0.08 per cent AAG was dependent on vancomycin concentrations; the values ranged from 21.1 per cent at the vancomycin concentration of 20 pg ml-' to 5.30 per cent at 2400 pg ml-I. However, binding to 4 per cent HSA was relatively constant, 8.79 f 2-43 per cent over a vancomycin concentration range of 20-2400 pg ml-'. The values to 4 per cent HSA alone and 0.08 per cent AAG alone did not predict the greater binding of vancomycin in the presence of both proteins, especially at higher concentrations of vancomycin; the values to 4 per cent HSA with 0.08 per cent AAG were constant, 26.3 f 3.74 per cent, at the vancomycin concentration range of 20-2400 pg ml-l. This suggested an interaction between the proteins, which resulted in enhanced binding of vancomycin. The protein binding of vancomycin to 4 per cent HSA with 0.08 per cent AAG was not influenced by the different incubation temperatures (4", 22", and 37"), quantities of heparin (up to 40 units ml-') or AAG (up to 0.16 per cent), or buffers (isotonic phosphate buffer of pH 7.4, phosphate buffer of pH 7-4 and 0.9 per cent NaCl solution) at the vancomycin concentration of 80 pg ml-I. Vancomycin was found to be stable in human serum albumin or in isotonic phosphate buffer of pH 7.4.