By using whole-cell pH_i epifluorescence (AM-loaded carboxy-SNARF-1) we evaluated the functional coupling of the Na⁺-HCO₃^– cotransporter (NBC) and the Na⁺/H⁺ exchanger (NHE) with carbonic anhydrase (CA) in ventricular myocytes isolated enzymically from humanely killed Sprague-Dawley rats. We analysed the pH_i recovery from an acid-load (NH₄⁺ prepulse technique) in myocytes superfused with HCO₃^–/CO₂-buffered Tyrode in the presence of cariporide or S0859 to measure the flux mediated by NBC and NHE, respectively. Each set of experiments was carried out in the presence or absence (control) of acetazolamide (ATZ; 100μM), a membrane-permeant CA inhibitor, in order to determine whether the activities of these transporters are functionally coupled to CA. NHE-mediated flux was not affected by ATZ (n=17). NBC showed a tendency towards lower flux in the presence of ATZ (n=26), particularly at lower pH_i values but without reaching significance. In order to evaluate the effect of ATZ under high NBC flux conditions and over a wider pHi range, we monitored NBC-mediated pH_i recovery in iso-osmotic solutions containing high [K⁺] (45mM). This resulted in a marked increase (3.38-fold) of NBC flux over the pH_i range 6.40-6.85. ATZ caused a significant reduction by 51% ± 2 of NBC activity (n=12, p<0.05; unpaired t test). CA activity in cardiac myocyte lysates was assayed in order to test ATZ potency. Cell lysates were obtained by resuspending ventricular myocytes in 20mM Hepes buffer (pH 8.0 at 4°C) and subsequent sonication. The time courses of pH change at 4°C after addition of 15.6mM CO₂ to lysates +/- 100μM ATZ, and to HEPES-buffer as blank were recorded. CO₂ hydration rates were significanly reduced in the presence of ATZ (n=6, p<0.05; unpaired t test) and were similar to the rate of the un-catalysed reaction, showing that ATZ effectively abolished CA activity in our assay. Our results show that CA activity does not enhance NBC-mediated acid efflux under normal low-flux conditions. However, under conditions of high NBC activity (e.g. hyperkalaemia combined with low pH_i) CA activity is required to sustain acid extrusion. The enzyme therefore provides a safety mechanism allowing high NBC flux under extreme conditions. This mechanism may be of relevance under conditions associated with acid-overload, such as post-ischaemic reperfusion of the myocardium.