The alpha-adrenergic stimulation by phenylephrine of lacrimal gland protein secretion was pharmacologically characterized. Acini, prepared from rat exorbital lacrimal glands, were incubated with agonists, antagonists or both for 0-20 min. Peroxidase secretion, an index of protein secretion, was measured spectrophotometrically. Peroxidase secretion was stimulated by the alpha 1-adrenergic agonists clonidine. The non-selective alpha-adrenergic antagonist phentolamine completely inhibited phenylephrine-induced secretion. The selective alpha 1-adrenergic alkylating agent phenoxybenzamine and the selective alpha 1-adrenergic antagonist prazosin partially inhibited phenylephrine-induced secretion. The alpha 2-adrenergic antagonist yohimbine, the beta-adrenergic antagonist timolol, and the dopaminergic antagonist haloperidol also inhibited phenylephrine-induced secretion but were 100-fold less potent than prazosin. It is concluded that phenylephrine activates an alpha 1-adrenergic pathway, but not an alpha 2-adrenergic, beta-adrenergic or dopaminergic pathway, to stimulate lacrimal gland protein secretion from acini.