We developed a multiple gas rebreathing technique for measuring lung diffusing capacity (DL(CO)), lung volume (V(L)) and cardiac output simultaneously in conscious spontaneously breathing small animals. Lung volume was measured from the dilution of methane (CH4) or sulfur hexafluoride (SF6) and verified independently by a helium washout technique. Cardiac output and DL(CO) were estimated from the uptake of acetylene and carbon monoxide, respectively. We tested guinea pigs at two levels of alveolar oxygen tension in order to estimate membrane diffusing capacity and pulmonary capillary blood volume by the Roughton-Forster technique. Results show that measured DL(CO) are consistent with reported values in anesthetized guinea pigs as well as with allometric comparison across species. Lung volume estimated from SF6 dilution agreed closely with that estimated independently from helium washout; however, lung volume estimated from CH4 dilution was systematically lower due to the addition of endogenously produced CH4 to the rebreathing system. We conclude that this technique can be used to measure resting lung function in conscious unsedated small animals.