Single-breath diffusing capacity of the lung for carbon monoxide (DLCO) increases as lung volume increases above functional residual capacity (FRC). However, the physiological mechanism responsible for this increase remains controversial. This volume dependence of diffusing capacity could reflect changing regional distribution of inspired air as lung volume increases rather than a change in capillary blood volume or surface area for gas exchange. We measured DLCO during breath holding and during rebreathing with a technique employed to mix respired gases throughout the lung thereby minimizing regional distribution differences. Measurements were made 1,500 ml above FRC and near total lung capacity (TLC). Breath holding DLCO was 18% higher near TLC than at 1,500 ml above FRC (P less than 0.05). Rebreathing DLCO was 16% higher near TCL than at 1,500 ml above FRC (P less than 0.01). Equality of results by the two techniques indicates that changes in DLCO with lung volume are not a consequence of the changing distribution of inspired air. Our results are compatible with the hypothesis that effective surface area of the lung increases as lung volume expands.