Background: Current methods of renal replacement therapy, combining convection and diffusion, are largely unsatisfactory in removing uremic toxins. Adsorption is a third mechanism that has been applied in extracorporeal therapy. This study evaluates the impact of hemodiafiltration with on-line regeneration of ultrafiltrate, a new two-step integrated sorbent system, on in vivo removal of a wide spectrum of solutes with different molecular weights. Methods: Pre- and post-dialysis concentrations of small, medium-size, and large molecules were determined in ten patients undergoing regular hemodiafiltration treatments with on-line regeneration of the ultrafiltrate. We also analyzed, at different times of the same dialysis session, the inlet and outlet ultrafiltrate; the latter had been regenerated by the sorbent cartridge and was used as reinfusion liquid. The mean dialysis time was 260 ± 21.2 min with a blood flow of 361 ± 33.3 ml/min and a reinjection volume of 3.6 ± 0.2 l/h. Results: Urea, creatinine and phosphate reduction ratio were respectively 69.8 ± 8.2, 61.9 ± 5.5, and 40.2 ± 17.3%. Removal of medium-size markers such as calcitonin, osteocalcin, β2-microglobulin, cystatin C, myoglobin and prolactin varied between 24 and 60%. The percentage of reduction for retinol binding protein and α1-microglobulin was negligible and we were unable to demonstrate any removal of α1-acid glycoprotein, pre-albumin, and albumin in the regenerated ultrafiltrate. Conclusion: The hemodiafiltration with on-line regeneration of ultrafiltrate is a new hemodialysis system, which allows uremic toxin removal over a wide molecular-weight spectrum.