BACKGROUND The tampering of athlete's urine samples by the addition of proteolytic enzymes during the doping control sampling procedure was reported recently. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in urine samples to chemically inactivate proteolytic enzymes and improve the electrophoteric signal of erythropoietin (EPO) in human urine. METHODS The stabilization mixture applied was a combination of antibiotics, antimycotic substances and protease inhibitors. A series of incubation experiments were conducted under controlled conditions in the presence and absence of the stabilization mixture in urine aliquots spiked with six proteases. Two different analytical techniques were applied for the qualitative and quantitative EPO measurement: isoelectric focusing (IEF) and chemiluminescent immunoassay respectively. RESULTS The addition of the chemical stabilization mixture into urine aliquots substantially improved EPO detection in the presence of proteolytic enzymes following incubation at 37 degrees C or storage at -20 degrees C. CONCLUSIONS The results of this study indicated that the stabilization of urine prior to the sample collection procedure with the proposed chemical mixture might prove to be a useful tool for the preservation of anti-doping samples.