Clenbuterol, a compound classified as a ₂-adrenoceptor (AR) agonist, has been employed in combination with left ventricular assist devices (LVADs) to treat patients with severe heart failure (HF). Previous studies have shown that chronic administration of clenbuterol affects cardiac excitation-contraction coupling. However, the acute effects of clenbuterol and the signaling pathway involved remain undefined. We investigated the acute effects of clenbuterol on isolated ventricular myocyte sarcomere shortening, Ca²⁺ transients and L-type Ca²⁺ current and compared these effects to two other clinically used ₂-AR agonists, fenoterol and salbutamol. 30 µM clenbuterol produced a negative inotropic response, whereas fenoterol showed a positive inotropic response. Salbutamol had no significant effects. Clenbuterol reduced Ca²⁺ transient amplitude and L-type Ca²⁺ current. Selective ₁-AR blockade did not affect the action of clenbuterol on sarcomere shortening but significantly reduced contractility in the presence of fenoterol and salbutamol (p<0.05). Incubation with 2 µg/ml pertussis toxin significantly reduced the negative inotropic effects of 30 µM clenbuterol. In addition, overexpression of G_i by adenoviral transfection induced a stronger clenbuterol-mediated negative inotropic effect, suggesting the involvement of the G_i protein. We conclude that clenbuterol does not increase and, at high concentrations, significantly depresses contractility of isolated ventricular myocytes, an effect not seen with fenoterol or salbutamol. In its negative inotropism clenbuterol predominantly acts through G_i, and the consequent downstream signaling pathways activation may explain the beneficial effects observed during chronic administration of clenbuterol in patients treated with LVADs.