Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed in specific areas of the developing central and peripheral nervous systems. We previously demonstrated that a membrane-bound and constitutively active form of the ALK protein tyrosine kinase (PTK) domain induced the neuron-like differentiation of PC12 cells through specific activation of the mitogen-activated protein kinase (MAP kinase) pathway. Its PTK domain had been originally identified in a nucleo-cytosolic and constitutively active transforming protein, NPM-ALK. Downstream targets involved in oncogenic proliferation and survival processes have been proposed to include phospholipase Cgamma (PLCgamma), phosphoinositide 3-kinase (PI 3-kinase)/AKT, STAT 3/5 and Src. We therefore postulated that activation of specific signaling pathways leading to differentiation or proliferation can be differently controlled depending on the subcellular localization of ALK PTK domain. To increase knowledge of its physiological role in the nervous system, we focused in the present study on the influence of its subcellular localization on neuronal differentiation. To achieve this goal, we characterized biological responses and transduction pathways in PC12 cells elicited by various constructs encoding membrane-bound (through transmembrane or myristyl sequences) or cytosolic ALK-derived proteins. In order to control the activation of their PTK domain, we used an inducible dimerization system. Here, we demonstrate that membrane attachment of the ALK PTK domain, in PC12 cells, is crucial for initiation of neurite outgrowth and proliferation arrest through a decrease of DNA synthesis. Furthermore, we show that this differentiation process relies on specific and sustained activation of ERK 1/2 proteins. By contrast, activation of the cytosolic form of this domain fails to induce MAP kinase activation and cell differentiation but promotes a PI 3-kinase/AKT-dependent PC12 cell proliferation. These data indicate that subcellular localization of the ALK PTK domain was a determinant for the control and specificity of downstream transduction cascades and was crucial for deciding the fate to which the neuronal cell will be committed.