The possible involvement of protein phosphorylation during beta-adrenergic receptor stimulation in rat submandibular gland was investigated in vitro using a dispersed cell preparation. (-)-Isoproterenol, a beta-adrenergic agonist, or dibutyryl cAMP stimulation was associated with an enhanced phosphorylation of three protein bands having apparent molecular weights of 34,000, 26,000, and 21,000, respectively. (-)-Propranolol, a beta-adrenergic antagonist, inhibited the phosphorylation of the three proteins during beta-adrenergic stimulation but not during dibutyryl cAMP stimulation. The three proteins were not fragments of a higher-molecular-weight protein. Subcellular fractionation using differential centrifugation, fractionation in an aqueous two-phase polymer system, and discontinuous sucrose gradient ultracentrifugation coupled with marker enzyme analysis indicated that all three proteins were enriched in the same subfractions: a heavy plasma membrane fraction and a fraction containing plasma membrane and Golgi membrane material. The extent of protein phosphorylation for all three proteins increased as a function of time and dose after beta-adrenergic stimulation. After 20 min of maximal beta-adrenergic stimulation, the addition of a beta-adrenergic blocker caused a time-dependent decrease in the 32P content of all three proteins. Pure cholinergic or pure alpha-adrenergic receptor stimulation had no effect on the 32P content of the three proteins. These data are consistent with a role for cAMP-mediated protein phosphorylation during mucin secretion from rat submandibular cells.