The Na⁺-HCO₃^- cotransporter (NBC) plays a key role in intracellular pH (pH_i) regulation in normal ventricular muscle. However, the state of NBC in non-ischemic hypertrophied hearts is unresolved. In this study we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure-overload created by suprarenal abdominal aortic constriction of 50 % for 7 weeks. Intracellular pH was measured in single cells using the fluorescent pH indicator BCECF. Real-time polymerase chain reaction (PCR) analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: a) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic-constriction, b) the onset of NBC up-regulation occurred within three days following constriction, c) normal and hypertrophied ventricles displayed regional differences in NBC expression, d) acid extrusion via NBC (J_NBC) was significantly increased in hypertrophied myocytes, e) although acid extrusion via Na⁺-H⁺ exchange was also increased in hypertrophied myocytes, the relative enhancement of J_NBC was larger, f) membrane depolarization markedly increased J_NBC in hypertrophied myocytes, and g) losartan, angiotensin II AT₁ receptor antagonist, significantly attenuated the up-regulation of both NBCs induced by three weeks of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na⁺ overload, which may render the ventricles more vulnerable to calcium overload during ischemia/reperfusion.