The effects of neomycin on voltage-activated Ca 2+ channels (VACCs) were studied by Ca 2+-dependent K +- and veratridine-evoked [ 3H]dopamine release from rat striatal slices. Neomycin (0.01–1 mM) concentration dependently reduced K +-evoked [ 3H]dopamine release (IC 50 ∼25 μM), producing ∼98% inhibition at 1 mM. Contribution of N-, P- and Q-type Ca 2+ channels to this neomycin-sensitive [ 3H]dopamine release was tested by the combined application of 100 μM neomycin and selective Ca 2+ channel blockers. The effects of neomycin combined with 1 μM of ω-conotoxin GVIA (N-type Ca 2+ channels) or with 100 nM of ω-conotoxin MVIIC (Q-type Ca 2+ channels) were additive, excluding involvement of N- and Q-type Ca 2+ channels. However, the combined effects of neomycin with 30 nM of ω-agatoxin-IVA (P-type Ca 2+ channels) were not additive, suggesting involvement of P-type Ca 2+ channels in neomycin-induced inhibition of [ 3H]dopamine release. On the other hand, veratridine-evoked [ 3H]dopamine release was shown to be mediated by Q-type Ca 2+ channels only. In addition, neither the inhibitor of sarcoplasmic reticulum Ca 2+-ATPase thapsigargin (500 nM) nor the blocker of sarcoplasmic reticulum ryanodine Ca 2+ channels ryanodine (30 μM) modulate veratridine-evoked [ 3H]dopamine release, suggesting no contribution of intracellular Ca 2+ stores. Neomycin (up to 100 μM) did not affect veratridine-evoked [ 3H]dopamine release, suggesting that intracellular Ca 2+ stores are not a prerequisite for the action of neomycin. Lack of inhibitory effect of neomycin is taken as additional indirect evidence for the involvement of P-type Ca 2+ channels. In conclusion, therapeutically relevant concentrations of neomycin preferentially block P-type Ca 2+ channels which regulate dopamine release in rat striatum. This block could be responsible for aminoglycoside-induced toxicity.