We have examined the effects of 4-aminopyridine (4-AP) on single ATP-dependent potassium channels in patches excised from frog skeletal muscle. 4-AP applied to the internal face of the membrane caused a flickery block. We could not detect any flickery block when 10 mM 4-AP was applied to the external surface of the membrane. The reduction in mean unitary current by internal 4-AP was consistent with 1:1 binding with a Kd of 3.3 mM at 0 mV. The block was voltage-dependent, increasing with depolarization with an effective valency of 0.57. Rate constants for blocking and unblocking by 4-AP were obtained by fitting beta functions to the distribution of current amplitudes. Both rate constants were voltage-dependent. At 0 mV they were 17 mM-1 ms-1 and 61 ms-1. Simulation of the block using these rate constants produced a flickery block very similar to that observed experimentally.