Recent evidence suggests that carbonic anhydrase (CA) IX in humans is under the regulatory control of hypoxia inducible factor and is over-expressed in certain cancers. However, little is known of its presence in non-mammalian vertebrates, or its physiological function in any vertebrate. The objective of this study was to examine and characterize the presence, distribution, induction by hypoxia and physiological function of CA IX in the zebrafish. Zebrafish CA IX was highly expressed in the eye, brain and gastrointestinal tract, and showed increased expression in the eye, brain and muscle in response to hypoxia (water PO₂ = 24 mm Hg). The hypothesis that increased CA IX expression during hypoxia would act to attenuate intracellular acidosis was then examined. Muscle intracellular pH (pH_i) decreased after 4 h of hypoxic exposure (from 7.15 ± 0.02 to 7.06 ± 0.01 pH units), and did not recover by 24 h. Manipulation of extracellular CA activity via intraperitoneal injection of either bovine CA or the selective extracellular CA inhibitor F3500 revealed that while increased CA activity could fully restore pH_i, removal of extracellular activity did not result in further acidosis. An exercise-induced acidosis was also attenuated in fish treated with bovine CA; however the increased extracellular CA expression resulting from hypoxia had no affect. These data suggest that although extracellular CA can potentially minimize the impact of hypoxia on muscle pH_i, the actual level of extracellular CA activity is likely insufficient to achieve this goal, even when enhanced by hypoxia-induced increases in CA IX expression.