Receptor-mediated changes in cytoplasmic calcium concentrations occur either through release from intracellular calcium stores or by the opening of channels in the plasma membrane, allowing influx of calcium from the extracellular fluid. Carbachol, a muscarinic receptor agonist, stimulated both calcium influx and inositol 1,4,5-trisphosphate (InsP3)-mediated intracellular calcium release in A9 fibroblast cells expressing a m3 muscarinic receptor clone. The calcium influx persisted even after pretreatment of cells with phorbol 12-myristate 13-acetate, which completely prevented the rise in inositol phosphates and intracellular calcium levels. The calcium influx was blocked by divalent cations but was not affected by inhibitors of voltage-dependent calcium channels or high potassium depolarization, indicating the presence of a receptor-operated and voltage-insensitive calcium channel in these cells. Calcium influx was not stimulated by the addition of cAMP analogs or arachidonic acid. To examine the possible involvement of G proteins in m3 receptor-activated calcium influx, two chimeric m2 and m3 muscarinic receptors were expressed in A9 cells in which the third cytoplasmic loop (the primary structural determinant in G protein coupling selectivity of muscarinic receptors) had been exchanged between the m2 receptor, which has no effect on calcium influx, and the m3 receptor. Calcium influx was found to be associated with a structural component of the m3 muscarinic receptor other than the third cytoplasmic loop.