Protein secretion from rat parotid acinar cells occurs in both the absence and presence of secretory agonists. Release takes place by four pathways that are distinguished by combined examination of their timing following biosynthetic labeling, their relative composition of salivary proteins, and their sensitivity to secretagogue stimulation. Following pulse-labeling with a radioactive amino acid, two unstimulated export pathways are detected--a constitutive-like pathway that is coupled to maturation of secretory granules and the later unstimulated exocytosis of secretory granules. In both cases, protein release is insensitive to secretory antagonists. Two regulated secretory pathways are also detected. The major regulated pathway comprises stimulated exocytosis of secretory granules and requires application of beta-adrenergic agonists (> or = 1 microM). A newly discovered minor regulated pathway resembles the constitutive-like pathway in secretory composition but requires low-dose stimulation by either beta-adrenergic or cholinergic agonists. The latter pathway may provide a significant component of basal secretion by the parotid gland during periods between meals.