Calcineurin, a Ca(2+)-regulated protein phosphatase, links myocardial Ca(2+) signaling with hypertrophic gene transcription. Calcineurin abundance increases in pressure-overload hypertrophy and may reduce agonist-mediated phospholamban (PLB) phosphorylation to underlie blunted beta-adrenergic receptor (beta-AR) responsiveness in hypertension. This hypothesis was tested by measuring the effects of calcineurin inhibition on changes in cardiac contractility caused by beta-adrenergic stimulation in spontaneously hypertensive rats (SHR). Female SHR (age: 7 mo) and age-matched female Wistar-Kyoto rats (WKY) were studied. Heart weight-to-body weight ratio (P < 0.01) and systolic blood pressure (P < 0.01) were greater in SHR compared with WKY and were associated with increased myocardial calcineurin mRNA (CnAbeta) and activity (P < 0.05). beta-AR stimulation with isoproterenol (Iso) increased calcineurin activity (P < 0.05) in both WKY and SHR hearts, and this activity was suppressed with cyclosporin A (CsA) treatment. In SHR, CsA improved left ventricular whole heart and isolated myocyte beta-AR responsiveness by normalizing PLB phosphorylation at Ser(16) and Thr(17) (P < 0.05). These CsA-induced, PLB-mediated effects were associated with an augmentation in cardiomyocyte peak Ca(2+) and a reduced rate (time constant of isovolumic pressure relaxation, tau) and magnitude of diastolic Ca(2+) during beta-AR stimulation. In conclusion, CsA normalized the blunted beta-AR responsiveness associated with hypertension, in part, by mitigating calcineurin activity while improving PLB phosphorylation and subsequent sarcoplasmic reticulum Ca(2+) regulation.