The cellular mechanisms of Wenckebach periodicity were investigated in single, enzymatically dissociated guinea pig ventricular myocytes, as well as in computer reconstructions of transmembrane potential of the ventricular cell. When depolarizing current pulses of the appropriate magnitude were delivered repetitively to a well-polarized myocyte, rate-dependent activation failure was observed. Such behavior accurately mimicked the Wenckebach phenomenon in cardiac activation and was the consequence of variations in cell excitability during the diastolic phase of the cardiac cycle. The recovery of cell excitability during diastole was studied through the application of single test pulses of fixed amplitude and duration at variable delays with respect to a basic train of normal action potentials. The results show that recovery of excitability is a slow process that can greatly outlast action potential duration (i.e., postrepolarization refractoriness). Two distinct types of subthreshold responses were recorded when activation failure occurred: one was tetrodotoxin- and cobalt-insensitive (type 1) and the other was sensitive to sodium-channel blockade (type 2). Type 1 responses, which were commonly associated with the typical structure of the Wenckebach phenomenon (Mobitz type 1 block), were found to be the result of the nonlinear conductance properties of the inward rectifier current, IK1. Type 2 sodium-channel-mediated responses were associated with the so-called "millisecond Wenckebach." These responses may be implicated in the mechanism of Mobitz type 2 rate-dependent block. Single-cell voltage-clamp experiments suggest that variations in excitability during diastole are a consequence of the slow deactivation kinetics of the delayed rectifier, IK. Computer simulations of the ventricular cell response to depolarizing current pulses reproduced very closely all the response patterns obtained in the experimental preparation. It is concluded that postrepolarization refractoriness and Wenckebach periodicity are properties of normal cardiac excitable cells and can be explained in terms of the voltage dependence and slow kinetics of potassium outward currents. The conditions for the occurrence of intermittent activation failure during diastole will depend on the frequency and magnitude of the driving stimulus.