The aim of the study is to study vancomycin serum concentration changes during on-line hemodiafiltration (OL-HDF) and determine whether administration of vancomycin during the last hour of OL-HDF provides therapeutic serum concentrations. Vancomycin was administered intravenously at a dose of 15 mg/kg to 17 chronic hemodialysis patients who were enrolled into two study groups (A and B). In group A patients (n = 11), vancomycin was administered immediately postdialysis. Forty-three hours later, a 4-hour OL-HDF session was performed. Blood samples (S) for vancomycin measurement were drawn before, during, and after the session. In group B patients (n = 6), vancomycin was administered during the last hour of a 4-hour OL-HDF session on Monday. Vancomycin serum concentrations were measured postdialysis, as well as before and after the next two dialysis sessions of the week. In group A patients, mean vancomycin concentrations were reduced by 50% (S(pre): 10.2 ± 1.4 μg/ml, S(post): 5.0 ± 0.9 μg/ml). Vancomycin dialysance was 74.6 ± 29.3 ml/min, while the volume of distribution was 0.42 L/kg. In group B patients, mean vancomycin concentrations before the beginning of Wednesday and Friday dialysis sessions were 3.1 ± 0.7 and 1.4 ± 0.4 μg/ml, respectively. Postdialysis administration of vancomycin followed by OL-HDF resulted in a 50% reduction of vancomycin serum concentrations. Administration during the last treatment hour of OL-HDF results in subtherapeutic vancomycin serum concentrations over the course of the subsequent inter- and intradialytic intervals.