In many teleost fish, catecholamines activate a red blood cell (RBC) Na+/H+ exchanger (βNHE), raising RBC intracellular pH to protect haemoglobin-O2 loading. The present study tested the hypothesis that RBC intracellular carbonic anhydrase (CA) contributes to this adrenergic response. The pH of rainbow trout (Oncorhynchus mykiss) blood was monitored continuously in vitro using blood flowing in a semi-closed loop or in vivo using an extracorporeal circulation. Addition or injection of isoproterenol activated the βNHE, causing blood pH to fall (in vitro ΔpH=−0.28±0.03 pH units, N=16; in vivo, −0.12±0.02 pH units, N=6). Both in vitro and in vivo, inhibition of RBC CA by acetazolamide significantly decreased the magnitude of the adrenergic response (in vitro, ΔpH=−0.22±0.02 pH units, N=16; in vivo, −0.02±0.01 pH units, N=6) as well as the rate of recovery of blood pH following the adrenergic response. These results support the hypothesis that RBC intracellular CA plays an important role in the RBC adrenergic response of rainbow trout, and fuel speculation that interspecific differences in RBC CA activity are associated with the magnitude of the RBC adrenergic response.