Multiple subtypes of voltage-gated calcium channels are coupled to transmitter release from central neurons; however, only N-type channels have been shown to play a role in autonomic neurons. The aim of the present study was to investigate potential roles for other channel subtypes in transmitter release from parasympathetic neurons in the mouse bladder using calcium channel toxins alone and in combination. Transmitter release was measured indirectly by recording the contraction of bladder dome strips in response to electrical stimulation of the neurons by single pulses or trains of 20 pulses at 1-50 Hz. omega-Conotoxin-GVIA (GVIA) and omega-conotoxin-MVIIC (MVIIC) inhibited contractions in a concentration-dependent manner, with IC50 values of approximately 30 and 200 nM, respectively, at low stimulation frequencies. omega-Agatoxin-IVA (agatoxin) alone did not have any significant effect up to 300 nM. Cumulative addition of the toxins demonstrated that 300 nM agatoxin had a significant effect after N-type channels were blocked with 100 nM GVIA. MVIIC (3 microM) reduced the contraction amplitude further. Testing the toxins on the cholinergic or purinergic component of the contraction separately showed that acetylcholine release depends primarily on N-type channels and, to a lesser extent, on P- and Q-type channels, whereas ATP release involves predominantly P- and Q-type channels. In conclusion, parasympathetic neurons in the mouse bladder, like central neurons, use multiple calcium channel subtypes. Furthermore, the release of the two main transmitters in these neurons has differing dependencies on the calcium channel subtypes.