The autonomic nervous system plays a regulatory role in the differentiation and growth of salivary glands, and in the expression of salivary specific genes. Cystatin S, a member of the evolutionarily conserved family 2 of the cysteine proteinase inhibitor superfamily, expressed in submandibular and parotid glands of rats during development, can be induced in adults by the beta-adrenergic agonist isopreterenol (IPR). It was shown previously that unilateral sympathectomy or bilateral parasympathectomy reduces IPR-induced cystatin S expression. The present experiments demonstrate that IPR-induced cystatin S gene expression in submandibular glands is reduced as early as 3 days post bilateral denervation of both branches of the autonomic nervous system. The reduction is nearly equal to that of either sympathectomy or parasympathectomy alone, suggesting that factor(s) in both sympathetic and parasympathetic fibers are simultaneously required for IPR-induced cystatin S gene expression.