With the on-line preparation of substitution fluid, an easy-to-operate and cost-effective alternative to conventional hemodiafiltration (HDF) has been realized. The continuous filtration of dialysis fluid, furthermore, allows high volumes of exchange. Microbial contamination and subsequently endotoxins, however, may be present in dialysis fluid, and thus the microbiological safety has become a pivotal issue. In this clinical study we evaluated the safety of the Fresenius Medical Care on-line HDF system which is based on a two-stage filtration of dialysis fluid with upstream DIASAFE and downstream on-line HDF filter. During the three-month study period we failed to detect germs or endotoxins in the substitution fluid. Augmented plasma interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) concentrations were found neither during the intradialytic period nor when pre-session values at study begin and study end were compared. In addition, changes in the anti-endotoxin core antibody levels and soluble CD14 (sCD14) concentration, or pyrogenic episodes were not observed. On-line HDF with DIASAFE and on-line HDF filter thus represents a safe treatment modality by effectively depleting dialysis fluid of cytokine-inducing substances.