The rate of pH decrease in dog blood after addition of CO2 (dissolved in saline) was measured in vitro with a combination pH electrode and fast-response amplifier. The pH change was found to have an apparent half time of 6.0 +/- 0.5 s at 38 degrees C, similar to that predicted by previous mathematical simulations on the basis of uncatalyzed CO2 hydration kinetics and the buffering characteristics of plasma. Measurements were also made in vivo in dogs by withdrawing blood rapidly from a carotid artery through a temperature-controlled chamber containing a pH electrode. When the flow was stopped, we measured the pH change which occurs in blood (after it leaves the lungs) as the slow dehydration of bicarbonate continues. The measured half time was 7.3 +/- 0.6 s, again agreeing well with predictions. Essentially the same half time was found when the direction of approach to chemical equilibrium was reversed by adding CO2 to the inspired air. The rate of the reactions could be increased markedly by hemolyzing the blood to release carbonic anhydrase into the plasma.