On-line hemodiafiltration (HDF) provides the largest amount of blood purification over a wide molecular weight spectrum achievable with present renal replacement therapies. When used with state of the art dialysis membranes and treatment systems, the biocompatibility of on-line HDF is as high as can presently be defined. From an economic perspective, the added cost of the ultrafilters used to prepare the substitution solution is balanced by the therapeutic benefits of HDF. For optimal HDF, the ultrafiltration rate must be maximized with respect to the blood flow rate. In on-line HDF systems, the excess volume ultrafiltered, approximately 20 to 30 liters per treatment, is automatically replaced, preferably in postdilution mode, by a substitution solution that is continuously generated by stepwise ultrafiltration of dialysate. When properly prepared, this fluid fulfills the quality demands of commercially available infusion solutions; that is, it can be referred to as sterile and pyrogen-free. The most important factors in preparing substitution solution are the quality of the water, of the concentrates, of the ultrafilters, and the microbiological status of the entire flow path. The clinical safety of substitution solution prepared on-line has been documented by long-term users of on-line systems. Results from clinical studies with on-line HDF confirm the overall increased clearance of solutes in relation to high-flux dialysis using the same membrane.