Serine proteases play important physiological roles through their activity at G-protein coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C isoforms play in the regulation of PAR₂-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short circuit current (Isc) responses to basolateral application of the selective PAR₂ activating peptide, SLIGRL-NH₂, were monitored as a measure of net electrogenic ion transport caused by PAR₂ activation. SLIGRL-NH₂ induced a transient Isc response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609) and PI3kinase (LY294002). Immunoblot analysis revealed the phosphorylation of both PLC and PLC following PAR₂ activation. Pretreatment of the cells with inhibitors of protein kinase C (PKC) (GF 109203X), PKC/1 (Gö6976), PKC (rottlerin) but not PKC (selective pseudosubstrate inhibitor) also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKC1, PKC and PKC, but not PKC or PKC, in membrane fractions following PAR₂ activation. Pretreatment of the cells with U73122, ET-18 or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKC translocation. Immunoblots revealed that PAR₂ activation induced phosphorylation of both cRaf and ERK1/2 via PKC. Inhibition of PKC1 and PI3K had only a partial effect on this response. We conclude that basolateral PAR₂-induced chloride secretion involves activation of PKC1 and PKC via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.