The possibility that the ml muscarinic receptor subtype can induce release of intracellular granules and transmitters was studied by transfecting a cultured mast cell line. RBL-2H3 cells, with the ml receptor gene. Comparisons were made between carbachol- and antigen-induced activation of various secretory responses. Like antigen, carbachol stimulated inositol phospholipid hydrolysis and release of arachidonic acid with concomitant dose-dependent secretion of granular contents. Carbachol also stimulated a biphasic increase in intracellular calcium, as measured by single cell fura-2 measurements. Although the kinetics of the carbachol-induced rise in intracellular calcium differed from that induced by antigen, they both utilized the same intracellular pool of calcium, and the second phase of the rise in intracellular calcium was dependent on extracellular calcium in both cases. Thus, the ml muscarinic receptor activates release of granules by a mechanism ostensibly similar to that of antigen.