Normal processing of Alzheimer's beta-amyloid precursor protein (APP) is markedly stimulated by phorbol esters, but the underlying mechanisms have yet to be fully understood. In this study, we observed that: (a) Phorbol 12,13-dibutyrate (PDBu)-stimulated APP secretion in cultured SH-SY5Y neuroblastoma and fibroblast cells was blocked by EGTA and calpain inhibitors in a concentration-dependent manner, but not by other protease inhibitors. (b) Secretion of fibronectin, another secretory protein tested for comparison, was enhanced by PBDu, but insensitive to calpain inhibitors. (c) PDBu stimulated intracellular calpain activity as measured by the hydrolysis of a fluorogenic calpain substrate. (d) PDBu also induced rapid proteolysis of two endogenous substrates of calpains, i.e., tau and microtubule-associated protein-2 (MAP-2) and the proteolysis was blocked by EGTA and calpain inhibitors. Taken together, these results suggest that stimulation of APP alpha-processing by PDBu is through a mechanism that involves the activation of Ca2+ and, most notably, calpain. The implications of the findings are discussed in relation to the regulatory mechanism of APP alpha-processing. Published by Elsevier Inc.