The single-breath diffusing capacity of the lung (DLCO) is a commonly performed pulmonary function test. The current American Thoracic Society (ATS) recommendations for reproducibility of DLCO measurements suggest that two measurements for the DLCO agree within 10% or 3 mL/min/mm Hg of the average value. The European Respiratory Society (ERS) recommends that two measurements should agree within 10%. The objectives of the present study were to examine whether the current reproducibility criteria were met in a general pulmonary function laboratory and to determine whether alternative criteria might be appropriate. Cross-sectional study. University-based pulmonary function laboratory. Patients referred for spirometry, helium lung volumes, and DLCO measurement. None. In a sample of 6,193 patients referred for clinical testing, 98.3% had two DLCO values that fulfilled the current ATS criteria for reproducibility. The coefficient of variation (CV) and the percentage difference between two repeat measurements were inversely associated with the baseline DLCO and the FEV(1). As the baseline DLCO (percentage of predicted) or FEV(1) (percentage of predicted) decreased, there was an increase in the CV and the percentage difference. In contrast, the absolute difference between repeat measurements was relatively stable irrespective of the baseline DLCO or FEV(1) values. Other patient factors, such as gender and race, were not associated with measurement variability. Using an absolute difference of 2 to 2.5 mL/min/mm Hg between two DLCO measurements as alternative criteria for reproducibility, 91.5% and 95.8% of the patient sample fulfilled these criteria, respectively. Reproducibility of the DLCO measurement is generally much better than current standards allow. Future standards should consider an absolute difference rather than a percentage difference criterion for DLCO reproducibility.