BACKGROUND: The 2005 American Thoracic Society/European Respiratory Society guidelines for single-breath diffusing capacity of the lung for carbon monoxide (D-LCO, recommend a weekly 1 biological control test and/or D-LCO simulator to detect instrument error drift. Very little has been published regarding the results of such a quality assurance program. Our aim was to analyze the long-term stability of a portable D-LCO instrument. METHODS: We used a new EasyOne Pro system and checked its accuracy using a D-LCO simulator with 2 reference gases (concentration A: carbon monoxide [CO] = 0.1% and helium = 6.52%; concentration B: CO = 0.08% and helium = 7.21 %) during the first 3 y of use in our large clinical laboratory. To detect instrument drift, a healthy woman (MSC), age 43 y old at baseline, tested herself every week during this period of time. RESULTS: More than 6,000 spirometry and 5,000 D-LCO maneuvers were done using this instrument for patients during these 3 y. There were no failures in the daily volume and flow checks or the CO and helium calibration checks performed automatically by the instrument. The differences between the simulator D-LCO and the measured D-LCO were -0.91 +/- 1.33 mL/min/mm Hg and -0.61 +/- 1.45 mL/min/mm Hg for concentration A and concentration B, respectively. The results of the 110 biological control tests were: mean 30.8 +/- 1.7 mL/min/mm Hg (95% CI 30.5-31.1), coefficient of variation of 5.4% in D-LCO, and repeatability of 2.5 mL/min/mm Hg. Only 4 measurements were outside +/- 3 mL/min/mm Hg (3.6%). Her mean alveolar volume was 4.2 +/- 0.25 L with coefficient of variation of 6.2%; her inspired volume was 3.05 +/- 0.14 L, and coefficient of variation = 4.5%. CONCLUSIONS: Measurements of D-LCO were stable over the 3-y period without any need for manual recalibration of the instrument. The biological control was as good as the D-LCO simulator to evaluate this kind of device in a long-term laboratory quality control program.