We investigated the differential role of protein kinase C (PKC) isoforms in the regulated proteolytic release of soluble amyloid precursor protein (sAPPalpha) in SH-SY5Y neuroblastoma cells. We used cells stably transfected with cDNAs encoding either PKCalpha or PKCepsilon in the antisense orientation, producing a reduction of the expression of PKCalpha and PKCepsilon, respectively. Reduced expression of PKCalpha and/or PKCepsilon did not modify the response of the kinase to phorbol ester stimulation, demonstrating translocation of the respective isoforms from the cytosolic fraction to specific intracellular compartments with an interesting differential localization of PKCalpha to the plasma membrane and PKCepsilon to Golgi-like structures. Reduced expression of PKCalpha significantly impaired the secretion of sAPPalpha induced by treatment with phorbol esters. Treatment of PKCalpha-deficient cells with carbachol induced a significant release of sAPPalpha. These results suggest that the involvement of PKCalpha in carbachol-induced sAPPalpha release is negligible. The response to carbachol is instead completely blocked in PKCepsilon-deficient cells suggesting the importance of PKCepsilon in coupling cholinergic receptors with APP metabolism.