Angiontensin II type 2 receptor (AT2) is upregulated in failing hearts, but its effect on myocytes contractile function is not known. We measured fractional cell shortening and [Ca²⁺]_i transients in LV myocytes derived from transgenic mice in which ventricle-specific expression of AT2 was driven by the MLC2v promoter. Confocal microscopy studies confirmed upregulation of AT2 in the ventricular myocytes and partial colocalization of AT2 with AT1. Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/L [Ca²⁺]_o, 25°C) and the study of contractile reserve at faster pacing rates (1-5 Hz) revealed Ca²⁺- dependent contractile dysfunction in myocytes from AT2 transgenic mice. The comparison of two transgenic lines suggested a dose-dependent relationship between the magnitude of contractile dysfunction and the level of AT2 expression. Second, the activity of the Na⁺/H⁺ exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to - adrenergic versus angiotensin II stimulation differed. Both lines showed impaired contractile response to - adrenergic stimulation. Angiotensin II elicited an increase in contractility and intracellular Ca²⁺ in wildtype myocytes. However, angiotensin II caused a negative inotropic effect in myocytes from AT2 transgenic mice. In contrast with - adrenergic response, the depressed response to angiotensin II was related to the level of AT2 overexpression. In addition, the depressed response to angiotension II was also present in myocytes from young transgenic mice prior to development of heart failure. Thus, the chronic overexpression of AT2 has the potential to cause Ca²⁺ dependent and pH dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to angiotensin II.