To identify a role for protein kinase C in lacrimal gland protein secretion, we incubated rat exorbital lacrimal gland acini in the ester 4-beta-phorbol 12, 13 dibutyrate (beta-phorbol dibutyrate), its inactive isomer 4-alpha-phorbol 12, 13 dibutyrate (alpha-phorbol dibutyrate), and the diacylglycerol analog 1,2-oleoyl acetylglycerol (OAG). We determined protein secretion by measuring the activity of peroxidase, a protein secreted by lacrimal gland acini. beta-phorbol dibutyrate, but not alpha-phorbol dibutyrate, stimulated peroxidase secretion in a concentration-dependent manner with 3 X 10(-8) M producing maximal secretion. OAG (10(-6) M) also stimulated peroxidase secretion. To determine whether muscarinic and alpha 1-adrenergic agonists activate protein kinase C, we added beta-phorbol dibutyrate (10(-7) M) simultaneously with carbachol (10(-5) M) or phenylephrine (10(-4) M); under both conditions, secretion was less than additive. Protein secretion in the presence of beta-phorbol dibutyrate (10(-7) M) and vasoactive intestinal peptide (VIP) (10(-8) M), the latter that acts through cAMP, was additive, and when the beta-phorbol dibutyrate but not the VIP concentration was decreased to 10(-8) M, secretion was potentiated. We conclude that muscarinic and alpha 1-adrenergic agonists, but not VIP, stimulated lacrimal gland protein secretion by activating protein kinase C.