Many hormonal, metabolic and enzymatic alterations have been described in patients with chronic renal failure (CRF) after prolonged hemodialysis treatment. This toxicity has been associated with the accumulation of middle molecular weight components, called ‘middle molecules’, in their blood. To investigate the chemical nature of these molecules, 20 CRF patients were studied and compared with 10 control subjects. Sera from these individuals were treated with molecular exclusion chromatography, the components of high molecular weight were eliminated from the elution peaks, and the remainder was then concentrated by lyophilization. The zones of greatest concentration were fractioned by ionic exchange chromatography and by HPLC in reverse phase. Infrared spectroscopy was also performed on the most relevant zones, as well as SDS/PAGE. Finally, the amino acid sequence of a peak presenting the best conditions was analyzed. The results indicate that the majority of the compounds isolated are peptides, amino acids or amino alcohols, and that many of these ‘middle molecules’ are joined to trace metals.