1. The blocking action of 4-aminopyridine (4-AP) on a delayed rectifier Kv1.2 K+ channel expressed in oocytes was investigated at room temperature (22 degrees C) and physiological temperature (34 degrees C) using the double-electrode voltage clamp and patch clamp techniques. 2. At room temperature, 4-AP (100 microM) inhibition occurred only after activation of current. The rate of onset of block was dependent upon the length of time current was activated by a depolarizing step. Similarly, removal of block required current activation. The degree of steady-state block by 4-AP was not reduced by increasingly more depolarized step potentials. The degree of steady-state block also did not change over the duration of a 1 s step. 3. When channels were nearly fully inactivated, 4-AP produced no additional block of a subsequent depolarizing step, suggesting that 4-AP did not bind when channels were in the inactivated state. In single channel experiments, 4-AP decreased the mean open time in a dose-dependent manner but did not alter the single-channel current amplitude. 4. At 34 degrees C the I-V relationship and inactivation curve shifted to more negative potentials. Increasing the temperature to 34 degrees C did not alter the degree of block by 4-AP, although the rate of onset of block was greatly enhanced. 5. Results suggest that 4-AP binds to the open state of the Kv1.2 channel and is trapped when the channel closes. 4-AP cannot bind when the channel is closed or inactivated prior to the addition of the drug. C-type inactivation and 4-AP binding to the channel are mutually exclusive. A model for the proposed mechanism of action of 4-AP on the Kv1.2 channel is proposed based on experimental data.