The neuronal endoplasmic reticulum (ER) contributes to many physiological and pathological processes in the brain. A subset of dendritic spines on hippocampal neurons contains ER that may contribute to synapse-specific intracellular signaling. Distribution of ER to spines is dynamic, but knowledge of the regulatory mechanisms is lacking. In live cell imaging experiments we now show that cultured hippocampal neurons rapidly lost ER from spines after phorbol ester treatment. ER loss was reduced by inhibiting -secretase (DAPT at 2 µM) and metalloproteinase (TAPI-0 and GM6001 at 4 µM) activity. Inhibition of protein kinase C also diminished loss of ER by preventing exit of ER from spines. Furthermore, -secretase and metalloproteinase inhibition, in the absence of phorbol ester, triggered a dramatic increase in spine ER content. Metalloproteinases and -secretase cleave several transmembrane proteins. Many of these substrates are known to localize to adherens junctions, a structural specialization with which spine ER interacts. One interesting possibility is thus that ER content within spines may be regulated by proteolytic activity affecting adherens junctions. Our data demonstrate a hitherto unknown role for these two proteolytic activities in regulating dynamic aspects of cellular ultrastructure, which is potentially important for cellular calcium homeostasis and several intracellular signaling pathways.—Ng, A. N., Toresson, H. -Secretase and metalloproteinase activity regulate the distribution of endoplasmic reticulum to hippocampal neuron dendritic spines.