The molecular and pharmacological characteristics of muscarinic receptor subtypes in the rat parotid acinar cell line, PAR-C5, were determined and compared with native rat parotid glands to evaluate the PAR-C5 cell line as a model to study receptor-mediated secretion. Reverse transcription-polymerase chain reaction (RT-PCR) identified mRNAs for M(3), M(4), and M(5) receptor subtypes in both PAR-C5 cells and parotid glands. Specific [N-methyl-(3)H]scopolamine binding in PAR-C5 and parotid membranes was to a single class of sites with mean K(D) values of 0.38 and 0.64 nM, respectively. Binding affinities (K(I) values) of muscarinic receptor subtype-selective drugs were obtained in side-by-side experiments comparing PAR-C5 cells with parotid glands. Nonlinear regression analysis indicated that competition binding curves for drugs in PAR-C5 cells and parotid glands fit best to a one-site binding model. K(I) values (nM) in PAR-C5 cells and parotid glands, respectively, for atropine (1.0, 2.1), darifenacin (1.2, 2.0), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (2.9, 2.4), tripitramine (220, 180), pirenzepine (320, 720), and methoctramine (1400, 1700) were consistent with their known affinities at the M(3) receptor subtype. Affinities (K(B) values) of muscarinic receptor subtype-selective drugs for blocking methacholine-stimulated Ca(2+) mobilization were determined to show which subtype mediates Ca(2+)-dependent secretion in Fura-2-loaded PAR-C5 cells. K(B) values (nM) for atropine (0.44), 4-DAMP (0.38), pirenzepine (140), and methoctramine (320) for blocking Ca(2+) responses correlated well with their known affinities at the M(3) receptor (r(2) = 0.99). These results show that at the level of mRNA, receptor protein and function, PAR-C5 cells and parotid glands are similar, establishing PAR-C5 cells as an important model for muscarinic receptor-mediated secretion.