We evaluated the role of myosin light-chain kinase (MLCK) and protein kinase C (PKC) in pepsinogen secretion from guinea pig gastric chief cells using a monolayer culture system of chief cells and an enzyme immunoassay system for guinea pig pepsinogen. An MLCK inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), significantly inhibited both the basal pepsinogen secretion and the secretion by carbamylcholine chloride (carbachol) or ionomycin without affecting intracellular free Ca2+ concentration ([Ca2+]i), but not by 12-O-tetradecanoylphorbol-13- acetate (TPA) or forskolin. A PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), significantly reduced the pepsinogen secretion by carbachol or TPA, but not by forskolin or ionomycin, and did not affect the basal secretion and the [Ca2+]i elevated by carbachol or ionomycin. We concluded that: (1) MLCK plays an important role in basal and drug-stimulated pepsinogen secretion, (2) MLCK is involved in the Ca(2+)-dependent intracellular pathway but not in the cyclic adenosine monophosphate (cAMP) dependent pathway, (3) PKC is irrelevant to activation of MLCK, and (4) increases in cAMP and [Ca2+]i are independent of activation of PKC.