Autoantibodies against the β₁-adrenoceptor (β₁-AAB) from patients with dilated cardiomyopathy (DCM) increase the beating frequency of cultured neonatal rat cardiomyocytes. This effect is accompanied by only a small increase in cAMP production. Here we have investigated whether β₁-AAB affect electrophysiological properties and cell shortening of isolated cardiomyocytes by interacting with theβ₁ -adrenoceptor. β₁-AAB were obtained during immunoadsorption of patients with DCM and were used for experiments in isolated myocytes cultured from neonatal rat hearts, or freshly isolated from adult rat ventricles or from human right atria. The unselective β -adrenoceptor agonist (−)-isoprenaline was studied for comparison. Immunoglobulin G(IgG) antibodies increased the spontaneous beating frequency of neonatal rat cardiomyocytes to a lesser degree than (−)-isoprenaline, but both effects were maximum and stable after 2 min. In rat ventricular and human atrial myocytes, IgG increased action potential duration (APD) in a concentration-dependent manner with larger effects on late than on early repolarization phases. Similar effects were obtained with purified β₁-AAB, whereas flow through of the chromatography column was ineffective. (−)-Isoprenaline prolonged APD to the same extent during plateau and late phase of repolarization. β₁-AAB increased L-Type Ca²⁺current in correspondence with the prolongation of APD. The effects of β₁-AAB and (−)-isoprenaline on APD were strongly attenuated after preincubation of the myocytes with the selective β₁-adrenoceptor antagonist (−)-bisoprolol. In addition, β₁-AAB increased cell shortening in ventricular myocytes from adult rat hearts.β₁ -AAB enhancing the beating frequency of cultured cardiomyocytes, increase L-Type Ca²⁺current, APD and contractility in freshly isolated cardiomyocytes mediated viaβ₁ -adrenoceptors. These effects may contribute to β₁-adrenoceptor-mediated cardiotoxicity in heart failure.