The effects of trachynilysin (TLY), a protein toxin isolated from stonefish (Synanceia trachynis) venom, were studied on the electrical and mechanical activities of frog atrial fibres. TLY (1 microg/ml) hyperpolarized the membrane, shortened the action potential (AP) duration (APD), exerted a negative inotropic effect and elicited contracture. These effects did not develop in the presence of atropine. TLY shortened the APD of fibres isolated from a frog completely paralyzed with botulinum type A toxin, in the presence of Ca2+ but not when Ca2+ was replaced by Sr2+. TLY increased the basal and the peak of the fluorescence ratio of stimulated fibres loaded with fura-2. Confocal laser scanning microscopy revealed the existence of a diffuse innervation in atrial tissue. Our results suggest that TLY enhances the release of acetylcholine from atrial cholinergic nerve terminals and activates indirectly muscarinic receptors leading to a shortening of APD. They also show that the mechanical effects induced by TLY are due to an increase of the Ca2+ influx and to a rise in intracellular Ca2+ levels which leads to (i) a slowing of the Na+/Ca2+ exchange activity, which accounts for the contracture and (ii) the activation of a Ca2+-dependent K+ current involved in the APD shortening.