Noninvasive techniques for assessing cardiopulmonary function in small animals are limited. We previously developed a rebreathing technique for measuring lung volume, pulmonary blood flow, diffusing capacity for carbon monoxide (DLCO) and its components, membrane diffusing capacity (DMCO) and pulmonary capillary blood volume (Vc), and septal volume, in conscious nonsedated guinea pigs at rest. Now we have extended this technique to study guinea pigs during voluntary treadmill exercise with a sealed respiratory mask attached to a body vest and a test gas mixture containing 0.5% SF6 or Ne, 0.3% CO, and 0.8% C2H2 in 40% or 98% O-2. From rest to exercise, O2 uptake increased from 12.7 to 25.5 ml.min(-1).kg(-1) while pulmonary blood flow increased from 123 to 239 ml/kg. The measured DLCO, DMCO, and Vc increased linearly with respect to pulmonary blood flow as expected from alveolar microvascular recruitment; body mass-specific relationships were consistent with those in healthy human subjects and dogs studied with a similar technique. The results show that 1) cardiopulmonary interactions from rest to exercise can be measured noninvasively in guinea pigs, 2) guinea pigs exhibit patterns of exercise response and alveolar microvascular recruitment similar to those of larger species, and 3) the rebreathing technique is widely applicable to human (similar to 70 kg), dog (20-30 kg), and guinea pig (1-1.5 kg). In theory, this technique can be extended to even smaller animals provided that species-specific technical hurdles can be overcome.