The mechanism of inward rectification was examined in cell-attached and inside-out membrane patches from Xenopus oocytes expressing the cloned strong inward rectifier HRK1. Little or no outward current was measured in cell-attached patches. Inward currents reach their maximal value in two steps: an instantaneous phase followed by a time-dependent "activation" phase, requiring at least two exponentials to fit the time-dependent phase. After an activating pulse, the quasi-steady state current-voltage (I-V) relationship could be fit with a single Boltzmann equation (apparent gating charge, Z = 2.0 +/- 0.1, n = 3). Strong rectification and time-dependent activation were initially maintained after patch excision into high [K+] (K-INT) solution containing 1 mM EDTA, but disappeared gradually, until only a partial, slow inactivation of outward current remained. Biochemical characterization (Lopatin, A. N., E. N. Makhina, and C. G. Nichols, 1994. Nature. 372:366-396.) suggests that the active factors are naturally occurring polyamines (putrescine, spermidine, and spermine). Each polyamine causes reversible, steeply voltage-dependent rectification of HRK1 channels. Both the blocking affinity and the voltage sensitivity increased as the charge on the polyamine increased. The sum two Boltzmann functions is required to fit the spermine and spermidine steady state block. Putrescine unblock, like Mg2+ unblock, is almost instantaneous, whereas the spermine and spermidine unblocks are time dependent. Spermine and spermidine unblocks (current activation) can each be fit with single exponential functions. Time constants of unblock change e-fold every 15.0 +/- 0.7 mV (n = 3) and 33.3 +/- 6.4 mV (n = 5) for spermine and spermidine, respectively, matching the voltage sensitivity of the two time constants required to fit the activation phase in cell-attached patches. It is concluded that inward rectification in intact cells can be entirely accounted for by channel block. Putrescine and Mg2+ ions can account for instantaneous rectification; spermine and spermidine provide a slower rectification corresponding to so-called intrinsic gating of inward rectifier K channels. The structure of spermine and spermidine leads us to suggest a specific model in which the pore of the inward rectifier channel is plugged by polyamines that enter deeply into the pore and bind at sites within the membrane field. We propose a model that takes into account the linear structure of the natural polyamines and electrostatic repulsion between two molecules inside the pore. Experimentally observed instantaneous and steady state rectification of HRK1 channels as well as the time-dependent behavior of HRK1 currents are then well fit with the same set of parameters for all tested voltages and concentrations of spermine and spermidine.