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ACh caused a transient increase in [Ca2+<\/jats:sup>]i<\/jats:sub>, and the increase was markedly inhibited by atropine or 4-diphenylacetoxy- N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the M3<\/jats:sub>subtype far exceeded that for either M1<\/jats:sub>or M2<\/jats:sub>as revealed by real-time quantitative PCR and in situ hybridization. The rise in [Ca2+<\/jats:sup>]i<\/jats:sub>evoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular Ca2+<\/jats:sup>deprivation. Caffeine had no effect on [Ca2+<\/jats:sup>]i<\/jats:sub>. ACh elicited Ca2+<\/jats:sup>-dependent K+<\/jats:sup>currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. 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