To avoid limitations associated with the use of single-breath and rebreathe methods for assessing the lung diffusing capacity for carbon monoxide (D(L)CO) during exercise, we developed an open-circuit technique. This method does not require rebreathing or alterations in breathing pattern and can be performed with little cognition on the part of the patient. To determine how this technique compared with the traditional rebreathe (D(L)CO,RB) method, we performed both the open-circuit (D(L)CO,OC) and the D(L)CO,RB methods at rest and during exercise (25, 50, and 75% of peak work) in 11 healthy subjects [mean age = 34 yr (SD 11)]. Both D(L)CO,OC and D(L)CO,RB increased linearly with cardiac output and external work. There was a good correlation between D(L)CO,OC and D(L)CO,RB for rest and exercise (mean of individual r2 = 0.88, overall r2 = 0.69, slope = 0.97). D(L)CO,OC and D(L)CO,RB were similar at rest and during exercise [e.g., rest = 27.2 (SD 5.8) vs. 29.3 (SD 5.2), and 75% peak work = 44.0 (SD 7.0) vs. 41.2 ml.min(-1).mmHg(-1) (SD 6.7) for D(L)CO,OC vs. D(L)CO,RB]. The coefficient of variation for repeat measurements of D(L)CO,OC was 7.9% at rest and averaged 3.9% during exercise. These data suggest that the D(L)CO,OC method is a reproducible, well-tolerated alternative for determining D(L)CO, particularly during exercise. The method is linearly associated with cardiac output, suggesting increased alveolar-capillary recruitment, and values were similar to the traditional rebreathe method.