We used fura-2 microfluorometry to examine the mechanism underlying the muscarinic receptor agonist-induced increase in the cytosolic Ca2+ concentration ([Ca]in) in acutely isolated chick ciliary ganglion neurons. The order of potencies of muscarinic agonists in increasing [Ca]in was: oxotremorine M > muscarine > methacholine > oxotremorine > bethanechol. The muscarine-induced increase in [Ca]in persisted after treatment with thapsigargin, which blocked caffeine- and muscarinic agonist-induced Ca2+ release. The muscarine-sensitive [Ca]in increase was inhibited by both L- and N-type Ca2+ channel blockers but potentiated by an L-type Ca2+ channel agonist. Muscarine was effective in increasing [Ca]in in the presence of a desensitizing concentration of nicotine, and simultaneous addition of maximal doses of muscarine and nicotine caused an additive increase in [Ca]in. On the other hand, nicotine-, ATP-, and high K(+)-induced increase in [Ca]in was markedly potentiated during continuous stimulation with muscarine. These results suggest that muscarinic receptor stimulation increases Ca2+ influx passing through voltage-dependent Ca2+ channels. However, the muscarine-induced Mn2+ influx was observed in only some muscarine-sensitive cells, suggesting that muscarine-induced depolarization is too weak to overcome the inhibitory effect of Mn2+ on Ca2+ channels.