The synthetic enkephalin analogue, [D-Ala2]Met-enkephalinamide (DALA) was used to investigate opioid peptide modulation of lacrimal protein secretion. By use of an in vitro perifusion system, the secretion of peroxidase by rat lacrimal gland fragments was measured during continuous stimulation for up to 60 min. DALA had no effect on unstimulated secretion of peroxidase. However, the addition of DALA to the perifusion medium resulted in a dose-dependent (10 nM-10 microM) inhibition of carbachol-stimulated peroxidase release. The maximum effect of DALA was achieved at a dose of 10 microM, which resulted in a 54% inhibition of carbachol-induced secretion. The opiate antagonist naloxone (10 nM-10 microM) did not alter basal or carbachol-stimulated peroxidase release. The effect on 10 microM carbachol-stimulated secretion by the addition of 3 microM DALA, however, was reversed in a dose-dependent manner by naloxone. The extent of inhibition of vasoactive intestinal peptidergic (VIPergic) stimulation (50 nM VIP) by DALA was similar to the inhibition of cholinergic stimulation of peroxidase release by gland fragments. Neither alpha 1-adrenergic stimulation of secretion nor a synergistic stimulation by VIP and carbachol was inhibited by the enkephalin analogue. We conclude that DALA exerts an inhibitory modulation of cholinergic and VIPergic stimulation of in vitro lacrimal protein secretion and suggest a physiological role for methionine enkephalin as an inhibitory peptide involved in the regulation of lacrimal gland function.