The single-breath diffusing capacity of the lung (DLCO) has been reported to decrease at a rate of 1.2 to 2.2% per hour during usual daytime hours. In an attempt to confirm these findings, we measured DLCO using both single-breath (DLCOs) and rebreathing techniques (DLCOR) in healthy, nonsmoking volunteers over the course of a day and again at different times on different days. Serial testing over a day showed a consistent fall (0.39%/h for DLCOS and 0.56%/h for DLCOR) in DLCO that was explained by increasing carboxyhemoglobin levels (COHb) and decreasing hemoglobin concentrations (Hb). When subjects were tested at different times on different days and compared using a paired t test, there was no change in DLCO over the time of day using either technique. We conclude that there is no diurnal variation in DLCO when adjusted for changes in Hb and COHb. The measurement of COHb levels and Hb concentrations for adjustment of DLCO results is important for correct interpretation of both individual and group studies of DLCO and should be performed whenever possible.