β-Amyloid precursor protein (APP) is a conserved and ubiquitous transmembrane glycoprotein strongly implicated in the pathogenesis of Alzheimer's disease but whose normal biological function is unknown. Analogy to the Notch protein suggests that APP is a cell-surface receptor that signals via sequential proteolytic cleavages that release its intracellular domain (AICD) to the nucleus. Because these cleavages are major targets for therapeutic inhibition, it is critical to elucidate their physiological function. AICD is stabilized by Fe65, interacts with the transcriptional factor Tip60, and translocates to the nucleus. Here, we show that endogenous AICD in primary neurons is detectable only during a short period of time during differentiation in culture. During this transient rise, a portion of AICD localizes to the nucleus. Subsequently, phosphorylation of the APP cytoplasmic domain at threonine 668 appears to disrupt the stabilizing interaction with Fe65 and thus downregulate AICD-mediated signaling. Furthermore, we find that the neuron-specific c-Jun N-terminal kinase JNK3, but not JNK1 or JNK2, mediates a substantial portion of this phosphorylation. We conclude that endogenous AICD undergoes tight temporal regulation during the differentiation of neurons and is negatively regulated by JNK3 via phosphorylation of APP at Thr668.