OBJECTIVE Lacritin protein is highly expressed in the lacrimal gland, secreted into tear fluid, and detected only in primates. The mechanism for lacritin secretion has not been fully investigated, because a system for culturing primate lacrimal acinar cells had not been established. The purposes of the present study were (1) to develop a procedure to culture lacrimal acinar cells from monkey and (2) to determine the mechanism for the secretion of lacritin in the culture system. METHODS Acinar cells from monkey lacrimal gland were cultured and characterized. Lacritin and other proteins were detected by immunohistochemistry, immunocytochemistry, and immunoblot analysis. Secreted proteins were also detected in the medium from stimulated acinar cells. mRNAs were determined by microarray and qPCR. Intracellular calcium levels were measured by calcium-4 assay. RESULTS Acinar cells cultured for 1 day contained adequate amounts of lacritin, lactoferrin, and lipocalin for use in lacritin secretion studies. The cholinergic agonist carbachol (Cch) stimulated the secretion of lacritin and increased intracellular Ca2+. Cch-induced lacritin secretion was inhibited by the store-operated calcium (SOC) channel inhibitor YM58483 and the PKC inhibitors GF109203 and Ro-32-0432. Cch-induced lacritin secretion was not inhibited by MAPKK inhibitor U0126, although p42/p44 MAPK was phosphorylated. Cch also enhanced gene transcription, which was inhibited by U0126, GF109203, and calcium chelators. CONCLUSIONS Successful culture of monkey lacrimal acinar cells showed that, among the prevalent tear proteins, the secretion of lacritin involved the PKC/Ca2+ pathway, not the p42/p44 MAPK pathway. Induction of transcription by Cch involved the independent p42/p44 MAPK and PKC pathways.