The single-breath (SB) method for determining the transfer factor for carbon monoxide (TLCO) is of limited value for the detection of diffusion disorders on the alveolar level, because the results are influenced by unequal distribution of ventilation and diffusion. The rebreathing method (RB) is thought not to be influenced by these inequalities. To the authors' knowledge, no study has measured both TLCORB and TLCOSB systematically and compared them with regard to the influence of unequal ventilation and diffusion. Therefore, the present study measured total lung capacity (TLC) as well as TLCO, both with the RB vital capacity method and the SB method, using the same apparatus in 10 healthy subjects and in 35 patients with chronic obstructive pulmonary disease (COPD). These patients are known to have increased unequal ventilation and diffusion in comparison with healthy subjects. In the healthy subjects, a small difference was found between TLC measured with the RB method (TLCRB) divided by the predicted value (TLCRB/pred) and TLCSB/pred (mean difference 0.07; SE = 0.02); no significant difference was found between TLCORB divided by the predicted value of TLCOSB (TLCORB/pred) and TLCOSB/pred. In the COPD patients, however, TLCRB/pred was larger than TLCSB/pred (mean difference 0.17; SE = 0.02) and TLCORB/pred was larger than TLCOSB/pred (mean difference 0.23; SE = 0.05). Multiple regression analysis revealed that in the COPD patients, 54% of the variance of the difference between TLCRB/pred and TLCSB/pred, and 76% of the variance of the difference between TLCORB/pred and TLCOSB/pred, were explained by parameters related to unequal ventilation and diffusion. In 25 of the 35 COPD patients, TLCOSB/pred was less than 0.8, whereas in 11 of these 25 patients, TLCORB/pred was more than 0.8. This difference was significant (P = 0.0005). In these 11 patients, the SB measurement resulted in the incorrect diagnosis of a diffusion disorder on the alveolar level. The RB method, however, never resulted in the diagnosis of a diffusion disorder when TLCOSB/pred was larger than 0.8. It is concluded that in a significant number of COPD patients, TLCOSB is below the normal range, whereas TLCORB is not below the normal range. This difference between TLCORB and TLCOSB is related to the combined effect of unequal ventilation and diffusion, and is of clinical importance for the detection of a diffusion disorder on the alveolar level.