Ca(v)2.1 (P/Q-type) channels possess a voltage-sensitive pore-forming alpha(1) subunit that can associate with the accessory subunits alpha(2)delta, beta and gamma. The primary role of Ca(v)2.1 channels is to mediate transmitter release from nerve terminals both in the central and peripheral nervous system. Whole-cell voltage-clamp studies in in vitro expression systems have indicated that accessory channel subunits can have diverse modulatory effects on membrane expression and biophysical properties of Ca(v)2.1 channels. However, there is only limited knowledge on whether similar modulation also occurs in the specific presynaptic environment in vivo and, hence, whether accessory subunits influence neurotransmitter release. Ducky, lethargic and stargazer are mutant mice that lack functional alpha(2)delta-2, beta(4) and gamma(2) accessory Ca(v) channel subunits, respectively. The neuromuscular junction (NMJ) is a peripheral synapse, where transmitter release is governed exclusively by Ca(v)2.1 channels, and which can be characterized electrophysiologically with relative experimental ease. In order to investigate a possible synaptic influence of accessory subunits in detail, we electrophysiologically measured acetylcholine (ACh) release at NMJs of these three mutants. Surprisingly, we did not find any changes compared to wild-type littermates, other than a small reduction (25%) of evoked ACh release at ducky NMJs. This effect is most likely due to the approximately 40% reduced synapse size, associated with the reduced size of ducky mice, rather than resulting directly from reduced Ca(v)2.1 channel function due to alpha(2)delta-2 absence. We conclude that alpha(2)delta-2, beta(4), and gamma(2) accessory subunits are redundant for the transmitter release-mediating function of presynaptic Ca(v)2.1 channels at the mouse NMJ.