Divalent cations and agents elevating cellular cAMP were tested for their effects on parathyroid hormone (PTH) secretion and protein kinase activity in dispersed bovine parathyroid cells. After incubation with secretagogue for 5–30 min, cells were sedimented, PTH in the supernatant was determined by RIA, and the pellet was disrupted by sonication for the measurement of protein kinase activity. Preliminary studies established conditions where the protein kinase activity ratio (AR = activity in the absence divided by that in the presence of 10^-6 M cAMP in the kinase assay) remained stable during the preparation and assay of cellular extracts. (−)Isoproterenol caused a rapid (within 5 min) increase in the AR from 0.28 to 0.63, which returned to 0.25–0.3 within 5 min after the addition of the potent βadrenergic blocker (β)propranolol. (β)Isoproterenol, dopamine, and the phosphodiesterase inhibitor methylisobutylxanthine caused parallel, dose-dependent increases in PTH release and the protein kinase AR of 2- to 2.5-fold. Calcium and magnesium, on the other hand, despite causing 2- to 4-fold inhibition of secretion at 2 and 5 mM, respectively, had no effect on the AR. Calcium (2.0 mM) likewise had only a modest (0–25%) inhibitory effect on the isoproterenol-stimulated increase in the AR in spite of a 3- to 4-fold inhibition of agonist-stimulated secretion. These results suggest that the stimulation of secretion associated with agents that elevate cAMP is mediated by cAMP. Changes in the degree of activation of protein kinase, on the other hand, cannot account quantitatively for the effects of divalent cations on basal or agonist-stimulated secretion.