The extent of activation of rat submandibular gland cyclic AMP-dependent protein kinase (EC 2.7.1.37) was determined in vitro using dispersed cells to assess the involvement of this enzyme in submandibular mucin secretion. cAMP-dependent protein kinase activation, as determined by activity ratio method, was markedly increased following beta-adrenergic receptor activation. 0.5 M NaCl was required in the homogenization buffer for stabilization of the hormonally activated cAMP-dependent protein kinase. A role for cAMP-dependent protein kinase activation in regulating mucin secretion was strongly suggested by the following: (1) the kinase activity ratio increased rapidly after beta-adrenergic receptor stimulation; (2) dose-response relationship of the kinase activation following beta-adrenergic receptor activation correlated with isoproterenol induced mucin release; (3) termination of beta-adrenergic mediated mucin secretion caused a rapid decrease in the kinase activity ratio; (4) dibutyryl cyclic AMP stimulation caused an increase in the kinase ratio; whereas (5) pure cholinergic and pure alpha-adrenergic receptor stimulation had no effect on endogenous kinase activity. Although cAMP-dependent protein kinase activation may not be the only regulator of mucin secretion, these data suggest an important regulatory role for this kinase activation during rat submandibular mucin release.