Abstract. The effects of acetylcholine (ACh) on the free intracellular calcium concentration ([Ca2+]i) of microdissected glomeruli were investigated using fura-2 fluorescence digital imaging and two-photon confocal microscopy. ACh caused a concentration-dependent [Ca2+]i increases with an initial peak followed by a sustained plateau, which was suppressed by reduced extracellular Ca2+ concentrations. The [Ca2+]i plateau was not affected by the L-type Ca2+ channel blocker nicardipine, whereas gadolinium and lanthanum (both at 1 μM) blocked the plateau. Diphenylacetoxy-N-methylpiperidine methiodide (100 nM), an M3/M5 receptor antagonist, and pirenzepine (1 μM), an M1 receptor antagonist, completely inhibited the effect of ACh. [Ca2+]i measurements using two-photon excitation of fluo-3 and staining of the cells with calcein/acetoxymethyl ester, for observation of the capillary network together with the glomerular cells, showed that [Ca2+]i was increased in single podocytes. Immunohistochemical studies did not demonstrate M3 receptor expression in glomerular cells. M1 receptors could be detected only in the parietal sheet of Bowman's capsule, whereas M5 receptors were found only in podocytes. The data show that ACh increases [Ca2+]i in podocytes of intact glomeruli, most likely via muscarinic M5 receptors.