The influence of adrenergic and cholinergic mediators on phospholipid secretion in rat sublingual salivary gland cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline-containing phospholipids over 30 min period averaged 1.93% of the total cellular labeled phospholipids in the absence of any mediator, and was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonists, pilocarpine and carbachol. A 2.9-fold increase in phospholipid secretion occurred with isoproterenol, while pilocarpine and carbachol evoked only 1.3-fold increase. The effect of isoproterenol was inhibited by alprenolol and that of pilocarpine and carbachol by atropine. In contrast to pilocarpine and carbachol, the enhanced phospholipid secretion due to isoproterenol was accompanied by an increase in cAMP concentration. The secretion of phospholipids was also stimulated by dibutyryl-cAMP and the protein kinase C activator, phorbol myristate acetate, but not by 4 alpha-phorbol 12,13-didecanoate which does not activate protein kinase C. Furthermore, the effects of dibutyryl-cAMP and phorbol myristate acetate were additive. The phospholipids secreted in response to isoproterenol exhibited a 52% decrease in lysophosphatidylcholine, while those secreted in response to pilocarpine and carbachol showed a 21-23% lower content of phosphatidylcholine, and were enriched in lysophosphatidylcholine (2.6-2.8-fold) and sphingomyelin (1.5-1.6-fold). The results indicate that salivary phospholipid secretion remains mainly under beta-adrenergic regulation, while the phospholipid makeup of the secretion is under cholinergic control.