Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of plasma membrane Na⁺/H⁺ exchanger (NHE1) and Cl^-/HCO₃^- exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H⁺ and HCO₃^-, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37±2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA expression two-fold, in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes, subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide, both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50=18 µM). ETZ and related CA inhibitor, methazolamide, prevented hypertrophy in adult cardiomyocytes. In addition, 6-ethoxyzolamide inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2±0.3 µM and 2.7±0.3 µM. PE significantly increased neonatal cardiomyocyte Ca⁺⁺ transient frequency from 0.33±0.4 Hz to 0.77±0.04 Hz following 24 h treatment; these Ca⁺⁺-handling abnormalities were completely prevented by 6-ethoxyzolamide (0.28±0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.