Rat parotid acinar cells dispersed by a combination of enzymatic treatments remain sensitive to adrenergic and cholinergic agonists. Previous studies have implicated Ca2+ in both adrenergic and cholinergic responses. This paper describes the effects of adrenergic and cholinergic stimulation upon 45Ca2+ fluxes in isolated parotid acinar cells. Suspensions of dispersed cells took up 45Ca2+ from the medium. The net rate of isotope influx was increased by the adrenergic agonists epinephrine, norepinephrine, isoproterenol, and phenylephrine, and by the cholinergic agonists acetylcholine and carbamylcholine. In 1 mM Ca2+, epinephrine was capable of increasing the 45Ca2+ influx in 40 min to three times that of resting cells. Isoproterenol, a beta-adrenergic agonist, was only half as effective as epinephrine in stimulating maximal calcium uptake although it was equally effective in stimulating maximal amylase release in the same cells. Experiments with the alpha-adrenergic antagonist phentolamine, the beta-adrenergic antagonist propranolol, and the cholinergic antagonist atropine confirmed that alpha- and beta-adrenergic and cholinergic stimulation each had a direct stimulatory effect on 45Ca2+ uptake. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate also caused some stimulation of net calcium uptake. Direct measurement of Ca2+ efflux indicated that the increased calcium uptake in the presence of epinephrine was not the indirect result of a decrease in efflux. The rates of both basal and epinephrine-stimulated calcium uptake increased with increasing calcium concentration in the medium. Epinephrine had little effect on the rate of calcium uptake at 0.15 mM Ca2+. Although the energy poison NaCN had little effect on the basal rate of calcium uptake, the stimulable component of calcium uptake was inhibited by NaCN at all calcium concentrations tested (0.2 to 4.1 mM).