Light and electron microscopic immunocytochemistry, in situ hybridization and Dot Blot analysis revealed intracellular localization of prolactin-like molecules and prolactin mRNA in epithelial cells of the lacrimal glands of rabbits. There was also positive immunostaining for prolactin receptors on acinar cells and some interstitial cells. On Western blots of homogenates of whole lacrimal gland, isolated lacrimal acinar cells, isolated lacrimal interstitial cells and peripheral blood lymphocytes, prolactin antibody consistently labeled protein bands migrating at approximately 36 and 50 kD. These data confirm that lacrimal gland acinar cells produce endogenous prolactin-like molecules, but also express prolactin receptors. Since prolactin immunoreactivity has been detected in tear fluid and we found no accumulations of immunogold label in endocytic or transport vesicles, we hypothesize that the prolactin-like molecules in tear fluid originate primarily from synthesis within the acinar cells. We hypothesize further that prolactin from pituitary and other non-acinar cell origin has a modulating influence on acinar cell activity as well as immune function in the lacrimal gland, and that some of the prolactin-like molecules produced by the acinar cells contribute to these functions by autocrine/paracrine mechanisms.