In adipocytes, stimulation of glucose transport by insulin is mediated largely by translocation of the GLUT4 isoform of glucose transporters from an intracellular store to the plasma membrane. Most endomembrane compartments are endowed with H(+)-pumping ATPases, and the resulting luminal acidification is thought to play a role in vesicular traffic. Chloroquine (Clq), a permeant weak base, was used to test whether endomembrane pH is an important factor in GLUT4 translocation. Under conditions chosen to optimize Clq uptake, the weak base precluded insulin-induced GLUT4 translocation and the associated stimulation of glucose transport. Clq also effectively dissipated the delta pH of acidic endomembrane compartments, assessed fluorimetrically. To define whether the intracellular GLUT4 storage compartment is acidic, immunoadsorption and immunoblotting experiments were performed to determine whether glucose transporters and vacuolar-type H+ pumps coexist in the same membranes. Unexpectedly, H+ pumps were not detectable in vesicles bearing GLUT4. Moreover, dissipation of endomembrane delta pH by monensin failed to inhibit insulin-stimulated GLUT4 translocation and hexose transport. Finally, the inhibitory effect of Clq persisted in the presence of monensin. We conclude that GLUT4 resides in an intracellular compartment devoid of H+ pumps. The insertion of this compartment into the plasmalemma is not regulated by transmembrane pH gradients. Clq impairs the stimulation of glucose transport by blocking translocation of GLUT4 by a pH-independent mechanism. Clq may provide a useful tool to elucidate the signalling or fusion steps involved in insulin-induced GLUT4 translocation.