Sixty-two serum concentrations were obtained from 12 infected patients enrolled in a vancomycin pharmacokinetic study. Both unbound and total serum vancomycin concentrations were measured using ultrafiltration and a commercial fluorescent polarization immunoassay. Ultrafiltrates were obtained by centrifugation at 1000 X g for ten minutes at room temperature and their assay indicated a range in protein binding from 7.9 to 71 percent. The mean protein binding (mean +/- SD) was 41.95 +/- 14.15 percent. No measurable adsorption of vancomycin onto the ultrafiltration membrane was noted. Orthogonal regression of unbound versus total vancomycin concentrations was described by the equation y = 0.597x-0.362 with a correlation coefficient of 0.948.