Summary The single exhalation analysis of carbon monoxide, acetylene, and methane allows the determination of intrabreath (regional) D L, pulmonary capillary blood flow and ventilation inhomogeneities during rest and exercise. We reasoned that this technique might be more sensitive in detecting regional pulmonary capillary abnormalities than resting single breath DL (DL sb). We selected a group of breast cancer patients in high-dose chemotherapy (HDCT) protocols who were at risk for pulmonary injury. We grouped the patients into pre-HDCT and post-HDCT, and used resting DL sb to further categorize the latter into those with and without pulmonary injury. We found that exercise DL increases were blunted in post-HDCT patients with low resting DL sb. More importantly, even in post-HDCT patients with normal resting DL sb, exercise DL response was reduced in the slowest emptying lung units along with evidence for ventilation inhomogeneities (increased methane slope). We conclude that exercise assessments of DL at low lung volumes and gas mixing properties may be sensitive indicators of lung injury.