The heart’s intensive pumping activity is powered by a uniquely high rate of mitochondrial respiration. This generates vast quantities of the waste-product CO2, which must be removed efficiently from mitochondria. Carbonic anhydrase (CA) enzymes, expressed at various sites in ventricular myocytes, may affect mitochondrial CO2-clearance by catalyzing CO2 hydration (to H+ and HCO3-) thereby changing the gradient for CO2 venting. In this study, we measured CA activity in cardiac myocytes, and investigated the effects of CA inhibition on cardiac energetics and function. CA activity in the cytoplasm of isolated ventricular myocytes was measured by fluorescence imaging of cells loaded with the pH reporter dye, cSNARF1 (10 µM, AM-loading for 5 min). Rapid exposure to CO2-containing solution evoked CO2 entry and its intracellular hydration which was only modestly accelerated by CA activity (2.7±0.3[SEM]-fold above spontaneous kinetics determined in the presence of the broad-spectrum CA inhibitor acetazolamide, ATZ, 100 µM). A similar experiment performed on isolated ventricular mitochondria demonstrated negligible intra-mitochondrial CA activity. CA activity was also investigated in intact hearts by 13C magnetic resonance spectroscopy (MRS) from the rate of H13CO3- production from 13CO2 released specifically from mitochondria by pyruvate dehydrogenase-mediated metabolism of hyperpolarized [1-13C]pyruvate. Under these conditions, the steady-state between de novo production and capillary wash-out produces a gradient of 13CO2 from mitochondria to the sarcolemma. CA activity measured upon [1-13C]pyruvate infusion was 11.4±0.9 fold above spontaneous kinetics (i.e. four-fold higher than the cytoplasm-averaged value). A fluorescent CA-ligand (fluorescein-thioureido-homosulfanilamide) co-localized with the mitochondrial marker TMRE (Pearson’s coefficient 0.735±0.06), indicating that mitochondria are near a CA-rich domain. Based on immunoreactivity, this domain comprises the nominally cytoplasmic CAII and sarcoplasmic reticulum-associated CAXIV. Inhibition of extra-mitochondrial CA activity with ATZ acidified the matrix by up to 0.1 unit (pH 7.73±0.03 to 7.62±0.02 measured by fluorescence imaging of permeabilized myocytes and isolated mitochondria by flow cytometry), impaired cardiac energetics (phosphocreatine-to-ATP ratio decreased from 2.02±0.13 to 1.58±0.14, measured by 31P-MRS of perfused hearts) and reduced contractility (developed pressure in perfused hearts decreased by 18±5%). These data provide evidence for a functional domain of high CA activity around mitochondria to support CO2-venting, particularly during elevated and fluctuating respiratory activity. Aberrant distribution of CA activity may therefore reduce the heart’s energetic efficiency.