Plasma pH has been postulated to change slowly in blood leaving the pulmonary capillaries because of the uncatalyzed dehydration of CO2. If so, there could be a difference between in vivo and in vitro arterial pH, the magnitude of which would be dependent on the venoarterial pH difference (v-aDpH). We tested this hypothesis in anesthetized dogs by changing v-aDpH by airway CO2 loading and by comparing arterial pH measured in vivo by a rapidly responding intravascular pH electrode with that measured in vitro by a conventional glass electrode. Using a multiple regression analysis, we found a small but significant contribution of venous pH to in vivo arterial pH, with a regression coefficient of 0.0718 (P < 0.0001), suggesting a postcapillary increase of in vivo arterial pH. When carbonic anhydrase was inhibited by the administration of acetazolamide, the effect of venous pH on arterial pH was abolished, and a unique relationship between in vivo and in vitro arterial pH was established (regression coefficient 1.02; P > 0.05, comparison with unity). These results could be accounted for in a computer simulation of gas exchange among alveolus, plasma, and erythrocyte. We conclude that there exists a small but measurable postcapillary increase in arterial pH.