Protease-activated receptor-2 (PAR-2) is expressed in the salivary glands and is expected to be a new target for the treatment of exocrine dysfunctions, such as dry mouth; however, the salivary secretory mechanism mediated by PAR-2 remains to be elucidated. Therefore, mechanism of the PAR-2-mediated salivary secretion was investigated in this study. We found that a PAR-2 agonist peptide, SLIGRL-OH, induced salivary flow in vivo and dose-dependent increase in [Ca(2+)](i) submandibular gland (SMG) acinar cells in wild-type (WT) mice and mice lacking M(3) or both M(1) and M(3) muscarinic acetylcholine receptors (mAChRs), whereas secretions in PAR-2 knockout (PAR-2KO) mice were completely abolished. The saliva composition secreted by SLIGRL-OH was similar to that secreted by mAChR stimulation. Ca(2+) imaging in WT acinar cells and beta-galactosidase staining in PAR-2KO mice, in which the beta-galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca(2+) response were enhanced in mice lacking M(3) or both M(1) and M(3) mAChRs, in which mAChR-stimulated secretion and Ca(2+) response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M(3)-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca(2+)-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.