The involvement of calcium, ATP, and cyclic AMP-dependent protein kinase activity in the release of amylase from rat parotid glands was examined. Pretreatment of the glandular tissue in 11.25 mM Ca2+ medium potentiated the secretory responses to: dibutyryl cyclic AMP, elevation of the extracellular K+ concentration, reduction of the H+ concentration, La3+, and caffeine. Uncoupling of oxidative phosphorylation blocked release induced by dibutyryl cyclic AMP, K+, and reduction of H+, but had no effect on La3+, caffeine or tolbutamide-stimulated release. Inhibition of cyclic AMP-dependent protein kinase activity blocked only dibutyryl cyclic AMP-induced release and did not inhibit the responses to K+, reduction of H+ or caffeine. The loss of lactate dehydrogenase was used to access the integrity of the tissue during amylase release. No significant increase in the release of lactate dehydrogenase was observed during the secretory responses to: dibutyryl cyclic AMP, La3+, caffeine, or tolbutamide. Triton X-100 and ethanol increased the efflux of both amylase and lactate dehydrogenase. The differential involvement of Ca2+, ATP, and cyclic AMP-dependent protein kinase activity in amylase release induced by the various secretagogues suggests that three types of reactions are involved in the release of amylase.