BACKGROUND P/Q- and N-type voltage-gated calcium channels (VGCC) are the principal subtypes mediating synaptic vesicle (SV) exocytosis. Both the degree of isoflurane inhibition of SV exocytosis and VGCC subtype expression vary between brain regions and neurotransmitter phenotype. We hypothesised that differences in VGCC subtype expression contribute to synapse-selective presynaptic effects of isoflurane. METHODS We used quantitative live-cell imaging to measure exocytosis in cultured rat hippocampal neurones after transfection of the fluorescent biosensor vGlut1-pHluorin. Selective inhibitors of P/Q- and N-type VGCCs were used to isolate subtype-specific effects of isoflurane. RESULTS Inhibition of N-type channels by 1 μM ω-conotoxin GVIA reduced SV exocytosis to 81±5% of control (n=10). Residual exocytosis mediated by P/Q-type channels was further inhibited by isoflurane to 42±4% of control (n=10). The P/Q-type channel inhibitor ω-agatoxin IVA at 0.4 μM inhibited SV exocytosis to 29±3% of control (n=10). Residual exocytosis mediated by N-type channels was further inhibited by isoflurane to 17±3% of control (n=10). Analysis of isoflurane effects at the level of individual boutons revealed no difference in sensitivity to isoflurane between P/Q- or N-type channel-mediated SV exocytosis (P=0.35). There was no correlation between the effect of agatoxin (P=0.91) or conotoxin (P=0.15) and the effect of isoflurane on exocytosis. CONCLUSIONS Sensitivity of SV exocytosis to isoflurane in rat hippocampal neurones is independent of the specific VGCC subtype coupled to exocytosis. The differential sensitivity of VGCC subtypes to isoflurane does not explain the observed neurotransmitter-selective effects of isoflurane in hippocampus.