DescriptionAction potentials (APs) are travelling waves of electrical activity in neurons composed of a rapid increase in membrane potential called a depolarization, followed by a repolarizing phase returning the membrane to a resting potential. Although APs are often thought of as all-or-nothing events, this is not necessarily the case. The AP waveform is generated by voltage-gated sodium and potassium channels whose composition, density and activity vary between and within neurons based on the function and output of that neuron. In addition to signal propagation, the purpose of an AP is to initiate the cascade of neurotransmitter release, beginning with the opening of voltage-gated calcium channels activated by the AP. Here, we set out to better determine how AP waveforms affect calcium influx and subsequent neurotransmitter release. The patch clamp technique has emerged as the best method to measure and study macroscopic electrical activity in neurons. Presynaptic APs at most synapses in the brain are difficult to study due to the small size of most presynaptic terminals. However, the calyx of Held synapse in the mammalian auditory brainstem is large enough to allow patch clamp recordings. In mouse brain slices, perfused with sodium and potassium channel blockers, various iii voltage protocols were tested to determine how modulation of AP kinetics alter calcium activity. First, various depolarization and repolarization rates were studied with test pulses of equivalent stimulus duration at 1 ms, showing that a repolarization/depolarization ratio between 1.5 and 2.3 is optimal for eliciting calcium influx. Additionally, depolarizations that follow the AP were studied and found to have no appreciable effect on calcium activity within the physiological range for AP durations in this neuron. However, if the repolarization rate is sufficiently fast, these currents were found to significantly alter the timing and magnitude of calcium influx. Finally, the AP was shown to minimize calcium channel inactivation to promote consistent and reliable neurotransmitter release. These findings will serve as the starting point for future work performing simultaneous pre- and postsynaptic patch clamp recordings to study transmission. This work promotes a better understanding of how AP kinetics affect calcium channel activity and thus neurotransmission.