A close anatomical relationship between nerves containing substance P and calcitonin gene-related peptide (CGRP) and mast cells containing serotonin has been demonstrated in the rat lacrimal gland. This study investigates the potential for peptidergic regulation of lacrimal mast cells by examining the actions of substance P, CGRP and serotonin on protein and peroxidase secretion from isolated lacrimal segments and on substance P and CGRP to release serotonin from the lacrimal mast cells. Substance P, CGRP and serotonin evoked marked increases in total protein and peroxidase from the lacrimal. Sodium cromoglycate, a mast cell stabilizer, significantly reduced or blocked the secretory responses elicited by these agonists. Chromatographic analysis using electrochemical detection revealed that substance P, but not CGRP, augmented the release of serotonin from the gland. The substance P evoked peroxidase secretion and serotonin release was blocked by CGRP and by sodium cromoglycate. These results support a role for mast cells in the regulation of lacrimal secretion and suggest a novel regulatory interaction between substance P and CGRP in the control of lacrimal function through a neuro-immune interaction.