Effects of a series of novel inhibitors of calmodulin or protein kinases on amylase release were studied in rat parotid slices. Amylase release induced by a cholinergic agonist, carbamylcholine, was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, 1-(5-chloronaphthalen-1-sulfonyl)-1H-hexahydro-1, 4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor, and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of Ca(2+)-activated, phospholipid-dependent protein kinase (protein kinase C), while N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), an inhibitor of cyclic AMP-dependent protein kinase (protein kinase A), did not inhibit the release. On the other hand, amylase release induced by a beta-adrenergic agonist, isoproterenol, was inhibited only by H-8, but not by W-7, ML-9 or H-7. These results suggest that cholinergic stimulation evokes amylase release via the Ca(2+)-dependent system which involves calmodulin, MLCK and protein kinase C, while beta-adrenergic stimulation via the cyclic AMP-dependent system involves protein kinase A.