The diffusing capacity of the lung for carbon monoxide (DLCO) is an important index of lung function but is not easily measured in spontaneously breathing animals with small lung volumes. Our aim was to devise a simple rebreathing method that would allow us to make serial measurements of DLCO in spontaneously breathing lambs during their first month after birth. By adding He to the rebreathing gas mixture, we were also able to measure functional residual capacity (FRC), enabling us to normalize DLCO with respect to FRC. We have compared FRC measured by the rebreathing technique with that measured by a closed-circuit helium dilution method (FRCcc). Using the rebreathing method we found highly significant positive correlations between DLCO and body weight (r = 0.70, P < 0.001) and between FRC and body weight (r = 0.79, P < 0.001). There was no significant change in DLCO/FRC over the first postnatal month; the mean value was 8.1 +/- 0.6 mL/min/mmHg/mL. Rebreathing FRC was highly correlated with FRCcc (r = 0.88, P < 0.001), but was lower than FRCcc by about 18%. In normal lambs DLCO and FRC, but not DLCO/FRC, increased during the first month after birth, suggesting that the increase in DLCO parallels lung growth. We conclude that the modified rebreathing method is suitable for measuring DLCO in small uncooperative spontaneously breathing animals.