Alterations in voltage-dependent gating of A-type potassium channels by the Sh5 mutation were studied using macroscopic and single-channel recording techniques on cultured embryonic myotubes from Drosophila. The mutation increases the voltage required to activate and inactivate the A1 channel by approximately 20 mV and decreases the steepness of the voltage dependence of steady-state inactivation. Boltzmann fits to the prepulse inactivation curves have slopes of e-fold/3.5 mV for wild type and e-fold/5.0 mV for Sh5. A kinetic analysis of single Sh5 A1 channels revealed that the transition rates into and out of the open state are not significantly affected by the mutation. In contrast, the latencies until the channel opens following a voltage step are increased at low voltages. These alterations probably do not result from an offset of the transmembrane voltage sensed by the channel as might be expected for an alteration in the surface charge of the protein. By using a kinetic model developed for wild-type A1 channels, we show that an alteration in the amplitude and voltage dependence of the deactivation rate for each subunit (beta) can account for all of the alterations observed.