To determine if different stimuli cause secretion of different proteins in lacrimal gland fluid (LGF), rabbits were anesthetized and LGF collected under baseline conditions (with the local anesthetic proparacaine), with ocular reflexes present, and in response to arterial injection of the cholinergic agonist acetylcholine (ACh) or the peptide vasoactive intestinal peptide (VIP). Proteins in LGF were separated by nondenatured gradient polyacrylamide gel electrophoresis. Except for minor differences, the number, the approximate molecular weights, and the amounts were the same in LGF secreted in response to four different stimuli. We concluded that the different stimuli caused protein release either from the same secretory cells or from different populations of secretory cells with the same secretory proteins.