We examined blood and urine concentrations of repetitive doses of inhaled salbutamol in relation to the existing cut-off value used in routine doping control. We compared the concentrations in asthmatics with regular use of beta2-agonists prior to study and healthy controls with no previous use of beta2-agonists. We enrolled 10 asthmatics and 10 controls in an open-label study in which subjects inhaled repetitive doses of 400 microgram salbutamol every second hour (total 1600 microgram), which is the permitted daily dose by the World Anti-Doping Agency (WADA). Blood samples were collected at baseline, 30 min, 1, 2, 3, 4, and 6 h after the first inhalations. Urine samples were collected at baseline, 0-4 h, 4-8 h, and 8-12 h after the first inhalations. Median urine concentrations peaked in the period 4-8 h after the first inhalations in the asthmatics and between 8-12 h in controls and the median ranged from 268 to 611 ng×mL (-1). No samples exceeded the WADA threshold value of 1000 ng×mL (-1) when corrected for the urine specific gravity. When not corrected one sample exceeded the cut-off value with urine concentration of 1082 ng×mL (-1). In conclusion we found no differences in blood and urine concentrations between asthmatic and healthy subjects. We found high variability in urine concentrations between subjects in both groups. The variability between subjects was still present after the samples were corrected for urine specific gravity.