The single-breath diffusing capacities for singly and doubly ¹⁸O-labeled CO₂, DL_{C¹⁶O¹⁸O} and DL_C¹⁸O2, as well as for NO, were determined in seven anesthetized rabbits to investigate whether the theoretically predicted ratio of specific blood uptake rates of both isotopic CO₂ species, _C¹⁸O2/_{C¹⁶O¹⁸O} = 2.0, can be derived from the measured values of DL_{C¹⁶O¹⁸O} and DL_C¹⁸O2. Data of DL were obtained by inflating the lungs with gas mixtures containing 0.35% C¹⁶O¹⁸O or 0.8% C¹⁸O₂ or 0.05% NO in nitrogen, with breath-holding periods of 0.05-0.5 s and 2-12 s for the CO₂ and NO tests, respectively. _C¹⁸O2/_{C¹⁶O¹⁸O} was calculated by applying the double-reciprocal Roughton-Forster equation to DL values obtained in each animal and by assuming that NO diffusing capacity represents the gas conductance of the alveolar-capillary membrane. The measured ratio was _C¹⁸O2/_{C¹⁶O¹⁸O} = 1.9 ± 0.2 (mean ± SD), thus comparing reasonably with the predicted one. Therefore, our findings provide evidence that the greater value of DL_C¹⁸O2 is mainly due to the twofold higher probability (or theta value) for C¹⁸O₂ than for C¹⁶O¹⁸O to disappear within red blood cells via isotopic exchange reactions.