The lacrimal gland is responsible for tear production, and a major protein found in tears is secretory component (SC), the proteolytically cleaved fragment of the extracellular domain of the polymeric immunoglobulin receptor (pIgR), the receptor mediating the basal-to-apical transcytosis of polymeric immunoglobulins across epithelial cells. Immunofluorescent labeling of rabbit lacrimal gland acinar cells (LGAC) revealed that the small GTPase rab3D, a regulated secretory vesicle marker, and the pIgR are colocalized in subapical membrane vesicles. In addition, the secretion of SC from primary cultures of LGAC was stimulated by the cholinergic agonist carbachol (CCH), and its release rate was very similar to that of other regulated secretory proteins in LGAC. In pull-down assays from resting LGAC, recombinant wild-type rab3D (rab3DWT) or the GDP-locked mutant rab3DT36N both pull down pIgR, but the GTP-locked mutant rab3DQ81L does not. When the pull-down assays are performed in the presence of GTP?S, GTP, or GDP?S, binding of rab3DWT to pIgR is inhibited. In blot overlays, recombinant rab3DWT bound to immunoprecipitated pIgR, suggesting that rab3D and pIgR may interact directly. Adenovirus-mediated overexpression of mutant rab3DT36N in LGAC inhibited CCH-stimulated SC release, and, in CCH-stimulated LGAC, pull-down of pIgR with rab3DWT and co-localization of pIgR with endogenous rab3D were decreased relative to resting cells, suggesting that the pIgR-rab3D interaction may be modulated by secretagogues. These data suggest that the novel localization of pIgR to the regulated secretory pathway of LGAC and its secretion therefrom may be effected by its novel interaction with rab3D.