Although an axoplasmic Ca²⁺ increase is associated with an exocytotic acetylcholine (ACh) release from the parasympathetic postganglionic nerve endings, the role of voltage-dependent Ca²⁺ channels in ACh release in the mammalian cardiac parasympathetic nerve is not clearly understood. Using a cardiac microdialysis technique, we examined the effects of Ca²⁺ channel antagonists on vagal nerve stimulation- and ischemia-induced myocardial interstitial ACh releases in anesthetized cats. The vagal stimulation-induced ACh release [22.4 nM (SD 10.6), n=7] was significantly attenuated by local administration of an N-type Ca²⁺ channel antagonist -conotoxin GVIA [11.7 nM (SD 5.8), n=7, P=0.0054], or a P/Q-type Ca²⁺ channel antagonist -conotoxin MVIIC [3.8 nM (SD 2.3), n=6, P=0.0002], but not by local administration of an L-type Ca²⁺ channel antagonist verapamil [23.5 nM (SD 6.0), n=5, P=0.758]. The ischemia-induced myocardial interstitial ACh release [15.0 nM (SD 8.3), n=8] was not attenuated by local administration of the L-, N-, or P/Q-type Ca²⁺ channel antagonists, by inhibition of Na⁺/Ca²⁺ exchange, or by blockade of inositol 1,4,5-triphosphate (IP₃) receptor, but was significantly suppressed by local administration of gadolinium [2.8 nM (SD 2.6), n=6, P=0.0283]. In conclusion, stimulation-induced ACh release from the cardiac postganglionic nerves depends on the N- and P/Q-type Ca²⁺ channels (with a dominance of P/Q-type) but probably not on the L-type Ca²⁺ channels in cats. In contrast, ischemia-induced ACh release depends on non-selective cation channels or cation-selective stretch activated channels but not on L-, N-, or P/Q type Ca²⁺ channels, Na⁺/Ca²⁺ exchange, or IP₃ receptor mediated pathway.