Aminoglycoside antibiotics can cause neuromuscular block by inhibiting Ca2+ influx into motor nerve terminals. P/Q-type Ca2+ channels, which are formed by alpha 1A subunits, are mainly responsible for depolarization-dependent presynaptic Ca2+ entry in motor neurons. We therefore investigated the possibility that aminoglycosides function as P/Q-type channel blockers. They inhibited [125I]-omega-CTx-MVIIC binding to P/Q-type channels in guinea pig cerebellum membranes with nanomolar IC50 values (e.g., 8 nM for neomycin). Divalent cations decreased the apparent affinity of neomycin. Barium inward currents through alpha 1A subunits expressed in Xenopus oocytes were partially blocked by therapeutic concentrations of aminoglycosides. This explains that therapeutically relevant concentrations of these drugs decrease the reserve of neuromuscular transmission, which can lead to neuromuscular block. We conclude that micromolar concentrations of aminoglycosides block not only N-type but also P/Q-type channels in mammalian neurons.