In guinea-pig ventricular myocytes, in which the deactivation of slowly activating delayed rectifier potassium current (IKs) is slow, IKs can be increased by rapid pacing as a result of incomplete deactivation and subsequent current accumulation. Whether accumulation of IKs occurs in dogs, in which the deactivation is much faster, is still unclear. In this study the conditions under which accumulation occurs in canine ventricular myocytes were studied with regard to its physiological relevance in controlling action potential duration (APD). At baseline, square pulse voltage clamp experiments revealed that the accumulation of canine IKs could occur, but only at rather short interpulse intervals (<100 ms). With action potential (AP) clamp commands of constant duration (originally recorded at rate of 2 Hz), an accumulation was only found at interpulse intervals close to 0 ms. Transmembrane potential recordings with high-resistance microelectrodes revealed, however, that at the fastest stimulation rates with normally captured APs (5 Hz) the interpulse interval exceeded 50 ms. This suggested that no IKs accumulation occurs, which was supported by the lack of effect of an IKs blocker, HMR 1556 (500 nM), on APD. In the presence of the beta-adrenergic receptor agonist isoproterenol (isoprenaline, 100 nM) the accumulation with AP clamp commands of constant duration was much more pronounced and a significant accumulating current was found at a relevant interpulse interval of 100 ms. HMR 1556 prolonged APD, but this lengthening was reverse rate dependent. AP clamp experiments in a physiologically relevant setting (short, high rate APs delivered at a corresponding rate) revealed a limited accumulation of IKs in the presence of isoproterenol. In conclusion, a physiologically relevant accumulation of IKs was only observed in the presence of isoproterenol. Block of IKs, however, led to a reverse rate-dependent prolongation of APD indicating that IKs does not have a dominant role at short cycle lengths.