The objective of this study was to evaluate the effects of lung perfusion on the slopes of phase III (S_III) and II (S_II) of a single-breath test of CO₂ (SBT-CO₂). 14 patients submitted to cardiac surgery were studied during the cardiopulmonary by-pass (CPB) weaning phase. Pump flow was decreased in 20 % steps, from 100 % (total CPB = 2.5 l min-1 m-2) to 0 % flow. This maneuver resulted in a progressive and opposite increase in pulmonary blood flow (PBF) while maintaining constant ventilation settings. SBT-CO₂, respiratory and hemodynamic variables remained unchanged before and after CPB reflecting a constant condition at those stages. S_III was similar before and after CPB (19.6 ± 2.8 and 18.7 ± 2.1 mmHg/L respectively). S_III was lowest during 20 % PBF (8.6 ± 1.9 mmHg/L) and increased in proportion to PBF increments until CPB exit (15.6 ± 2.2 mmHg/L, p < 0.05). Similarly, S_II and the CO₂ area under the curve increased from 163 ± 41 mmHg/L and 4.7 ± 0.6 ml at 20 % PBF to 313 ± 32 mmHg/L and 7.9 ± 0.6 ml (p < 0.05) at CPB end respectively. When SII and SIII were normalized by the mean expired CO₂ %, they remained unchanged during the protocol. In summary, the changes in PBF affect the slopes of the SBT-CO₂. Normalizing S_III and S_III eliminates the effect of magnitude of PBF on the shape of the SBT-CO₂ curve.