It is well known, that a non-selective β-adrenergic receptor agonist, the isoprenaline (ISO) activates several ionic currents in mammalian myocardium, however their relative contribution to the ISO-induced changes in action potential morphology is not well explored. Therefore, our aim was to describe the effects of ISO on action potential configuration, L-type Ca2+ current (ICa,L), slow delayed rectifier K+ current (IKs) and fast delayed rectifier K+ current (IKr) in canine left ventricular myocytes. Adult beagle dogs (n=39) were anaesthetized with the mixture of ketamine hydrochloride (10 mg/kg) and xylazine hydrochloride (1 mg/kg, both I.M.) according to a protocol approved by the local ethical committee, which conformed to the principles outlined in the Declaration of Helsinki. Single myocytes were obtained by enzymatic dispersion using the segment perfusion technique. Action potentials were recorded with conventional sharp glass microelectrodes, ionic currents were measured using conventional and action potential voltage clamp techniques on isolated ventricular cardiomyocytes (n=157) from dog hearts. Values are expressed as mean ± S.E.M., compared by ANOVA. ISO (10-100 nM) caused significant and reversible shortening of action potential duration accompanied by elevation of the plateau potential. Similar results were observed when ISO was applied after pretreatment with an IKr blocker (1 µM E-4031). In the presence of an IKs blocker (1 µM HMR1556) action potentials were significantly lengthened by ISO in spite of the pronounced plateau elevation. Both ISO-induced changes were prevented by pretreatment with nisoldipine (ICa,L blocker, 5 µM). Action potential voltage clamp experiments revealed a prominent slowly inactivating ICa,L followed by a rise in IKs. Similar ISO-induced responses were seen in the presence of 1 µM E-4031, while the ISO-induced activation of IKs was abolished by 1 µM HMR1556. Conventional voltage clamp experiments revealed that ISO increased IKs, IKr and ICa,L to 420±4 (n=5, p<0.05), 133±1 (n=8, p<0.05) and 340±13 % (n=8, p<0.05) of their baseline values, respectively, with the concomitant EC50 values of 14.5±1.1, 13.7±2.5 and 15.3±3.5 nM (n=5, n=8, n=8, respectively). Our results suggest that the ISO-induced activation of IKs – but not IKr – may be responsible for the observed shortening of action potentials in the canine myocytes. The similar EC50 values estimated for IKs, IKr and ICa,L may indicate a common mechanism responsible for the ISO-induced activation of these currents.