The effect of cholinergic agonists and antagonists on the intracellular free Ca2+ concentration ([Ca2+]i) of leech neuropile glial cells was investigated by use of iontophoretically injected fura-2. In neuropile glial cells, cholinergic agonists induced a marked increase in [Ca2+]i that was inhibited by d-tubocurarine, alpha-bungarotoxin, strychnine, and atropine. The efficacy of the various agonists and antagonists indicates that the [Ca2+]i increase is mediated by the nicotinic acetylcholine (ACh) receptors that have been characterized previously in these cells by using electrophysiological methods. In the presence of high agonist concentrations, [Ca2+]i partly recovered, suggesting that the ACh receptors desensitize. The [Ca2+]i increase induced by cholinergic agonists was abolished in Ca2(+)-free solution, which indicates that it is caused by Ca2+ influx from the external medium. The agonist-induced [Ca2+]i increase was partly preserved in Na(+)-free solution, whereas the agonist-induced membrane depolarization was strongly suppressed. The agonist-induced [Ca2+]i increase was also partly preserved in the presence of 5 mM Ni2+, which almost abolished the K(+)-induced [Ca2+]i increase mediated by voltage-dependent Ca2+ channels. It is concluded that at low agonist concentrations the [Ca2+]i increase in leech neuropile glial cells is mediated exclusively by the ion channels associated with the nicotinic ACh receptors. At high agonist concentrations, voltage-dependent [Ca2+]i increase in leech neuropile glial cells is mediated exclusively by the ion channels associated with the nicotinic ACh receptors. At high agonist concentrations, voltage-dependent Ca2+ channels activated by the concomitant membrane depolarization also contribute to the agonist-induced Ca2+ influx.