The time- and voltage-dependent properties of a slowly inactivating K+ current were investigated by using the single-electrode current- and voltage-clamp recording technique in CA3 hippocampal cells of organotypic slice cultures. After a period of prolonged hyperpolarization, the onset of action-potential discharge in response to depolarizing current injection was delayed by several seconds. The conductances underlying this delay were identified in voltage-clamp recordings. A biphasically decaying outward current was evoked when the membrane potential was stepped back to -60 mV after a 30 sec period of hyperpolarization. The fast component was identified as the previously described D-current and was blocked by 100 microM 4-aminopyridine (4-AP). The slow component, which we refer to as IK(slow), appeared to be mediated by K+ ions, because its reversal potential shifted in a Nernstian manner with changes in extracellular K+ concentration. It decayed with a time constant of 7.5 sec and required a hyperpolarizing prepulse below -95 mV for 5.5 sec for 50% recovery from inactivation. IK(slow) was found to be voltage-dependent, with 50% activation occurring at -65 mV and 50% steady-state inactivation occurring at -84 mV. It displayed minimal or no sensitivity to the K(+)-channel blockers 4-AP (0.1-5 mM), Cs+ (1 mM), tetraethylammonium (10-50 mM), Ba2+ (1 mM), dendrotoxin-alpha (5-10 microM), charybdotoxin (0.5-2.5 microM), or glibenclamide (5-10 microM) and was not affected by preventing increases in intracellular Ca2+ concentration with Ca2+ chelators. IK(slow) was reduced by activation of metabotropic glutamatergic and cholinergic receptors. In summary, the biophysical characteristics of IK(slow) suggest a role in determining discharge onset after a period of membrane hyperpolarization, and its modulation by G-protein-coupled receptors reveals an additional function for these receptors in the control of cellular excitability.