Measurements of CO(2) permeability in oocytes and liposomes containing water channel aquaporin-1 (AQP1) have suggested that AQP1 is able to transport both water and CO(2). We studied the physiological consequences of CO(2) transport by AQP1 by comparing CO(2) permeabilities in erythrocytes and intact lung of wild-type and AQP1 null mice. Erythrocytes from wild-type mice strongly expressed AQP1 protein and had 7-fold greater osmotic water permeability than did erythrocytes from null mice. CO(2) permeability was measured from the rate of intracellular acidification in response to addition of CO(2)/HCO(3)(-) in a stopped-flow fluorometer using 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) as a cytoplasmic pH indicator. In erythrocytes from wild-type mice, acidification was rapid (t((1)/(2)), 7.3 +/- 0.4 ms, S.E., n = 11 mice) and blocked by acetazolamide and increasing external pH (to decrease CO(2)/HCO(3)(-) ratio). Apparent CO(2) permeability (P(CO(2))) was not different in erythrocytes from wild-type (0.012 +/- 0.0008 cm/s) versus null (0.011 +/- 0.001 cm/s) mice. Lung CO(2) transport was measured in anesthetized, ventilated mice subjected to a decrease in inspired CO(2) content from 5% to 0%, producing an average decrease in arterial blood pCO(2) from 77 +/- 4 to 39 +/- 3 mm Hg (14 mice) with a t((1)/(2)) of 1.4 min. The pCO(2) values and kinetics of decreasing pCO(2) were not different in wild-type versus null mice. Because AQP1 deletion did not affect CO(2) transport in erythrocytes and lung, we re-examined CO(2) permeability in AQP1-reconstituted liposomes containing carbonic anhydrase (CA) and a fluorescent pH indicator. Whereas osmotic water permeability in AQP1-reconstituted liposomes was >100-fold greater than that in control liposomes, apparent P(CO(2)) (approximately 10(-3) cm/s) did not differ. Measurements using different CA concentrations and HgCl(2) indicated that liposome P(CO(2)) is unstirred layer-limited and that HgCl(2) slows acidification because of inhibition of CA rather than AQP1. These results provide direct evidence against physiologically significant AQP1-mediated CO(2) transport and establish an upper limit to the CO(2) permeability through single AQP1 water channels.