The influence of the mode of cell stimulation on the outward K+ current (I(o)) was studied in whole-cell patch-clamped human atrial myocytes. Acceleration of the rate of membrane depolarization at 1 Hz or during prolonged 5-s test pulses at 0.1 Hz increased the rate and extent of I(o) inactivation, resulting in enhanced inactivating (4.9+/-0.6 v 6.3+/-0.7 pA/pF) and suppressed maintained (5.9+/-1.2 v 3.2+/-0.3 pA/pF) current components. These alterations were associated with a leftward shift of the voltage-dependency of I(o), and persisted on returning to a control depolarization protocol (750-ms test pulses delivered at 0.1 Hz). The effects of increasing external K+ concentrations (40 m m) on the kinetics of I(o) were more pronounced following both rapid and prolonged depolarization (changes in I(t)/I(o)caused by 40 m m K+: 8.9+/-3.5% v 15.5+/-3.1% before and after prolonged depolarization; and 9.2+/-1.2% v 15.4+/-1.7% before and after rapid depolarization). The phosphatase inhibitor, okadaic acid, enhanced the effect of rapid and prolonged depolarization on I(o)whereas the inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMK-II) with KN-62 or KN-93, or by intracellular application of the autocamtide-2-related inhibitory peptide, suppressed it. In conclusion, rapid and prolonged membrane depolarization both cause a cumulative increase in the rate and extent of I(o)inactivation. This process involves slow potassium channel inactivation mechanisms, is regulated by CaMK-II, and may contribute to the electrical memory of the atrial myocardium.