We examined the subcellular distribution of a novel glucose transporter isoform (GLUT8) in murine N2A neuroblastoma cells. Exogenous expression of GLUT8-green fluorescent protein (GFP) DNA constructs mimicked the endogenous GLUT8 localization to intracellular vesicles and minimally to the Giantin-positive Golgi. This distribution was unlike the distributions of endogenous GLUT1 and GLUT3 (predominant neuronal isoform), which were limited predominantly to the plasma membrane and minimal in the cytoplasm. Although GLUT4-GFP (insulin responsive isoform) was expressed transiently, no endogenous GLUT4 was detected in N2A cells. By employing stable transfectants that expressed GLUT8-GFP, the effect of insulin and insulin-like growth factor-I, potassium chloride (depolarized state), and 3% oxygen on translocation of GLUT8 to the plasma membrane of N2A cells was examined immunohistochemically and by subfractionation, followed by Western blot analysis. None of these agents translocated GLUT8 to the plasma membrane. However, when the internalization dileucine motif (L12,13) of GLUT8 was mutated to a dialanine motif (A12,13), GLUT8 colocalized with GLUT3 in the plasma membrane. We conclude that GLUT8 translocation to the N2A cellular plasma membrane is not observed secondary to the various stimuli investigated. Mutation of the N-terminal dileucine motif led to constitutive GLUT8 localization in the plasma membrane. The endogenous stimulus required for translocating neuronal GLUT8 is unknown. This stimulus, which is necessary for uncoupling the “cytoplasmic vesicular anchor” of GLUT8, would be crucial for its glucose-transporting function. © 2004 Wiley-Liss, Inc.