We investigated the differential role of protein kinase C (PKC) isoforms in the regulated proteolytic release of soluble amyloid precursor protein (sAPPα) in SH-SY5Y neuroblastoma cells. We used cells stably transfected with cDNAs encoding either PKCα or PKCη in the antisense orientation, producing a reduction of the expression of PKCα and PKCη, respectively. Reduced expression of PKCα and/or PKCη 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 PKCα to the plasma membrane and PKCη to Golgi-like structures. Reduced expression of PKCα significantly impaired the secretion of sAPPα induced by treatment with phorbol esters. Treatment of PKCα-deficient cells with carbachol induced a significant release of sAPPα. These results suggest that the involvement of PKCα in carbachol-induced sAPPα release is negligible. The response to carbachol is instead completely blocked in PKCη-deficient cells suggesting the importance of PKCη in coupling cholinergic receptors with APP metabolism.