To investigate the nature of the muscarinic receptors present on parietal cell membranes, binding studies and polymerase chain reaction (PCR) amplification of parietal cell messenger (m) RNA were undertaken. Displacement of N-[3H]methylscopolamine by various muscarinic antagonists showed displacement with a single affinity. The apparent dissociation constant values were as follows: atropine (nonselective), 1.95 +/- 0.28 nmol/L; pirenzepine (M1), 169 +/- 24 nmol/L; AF-DX 116 (M2), 1542 +/- 33 nmol/L; and hexahydrosiladifenidol (M3), 29 +/- 3.4 nmol/L. These data confirmed the existence of only an M3 receptor linked to acid secretion as defined pharmacologically. PCR amplification of parietal cell mRNA with primers designed for detection of all known muscarinic receptor subtypes showed that only m3 fragments were produced from parietal cell mRNA, whereas m1 and m2 products could be detected in brain or cardiac mRNA. The m3 nature of the PCR product was confirmed by Southern blotting with 32P-labeled human m3 complementary DNA. Hence the two carbachol affinities and the separable cellular responses following muscarinic activation are caused by separate coupling pathways of the M3 receptor.