Phagosomes employ lytic enzymes, cationic peptides and reactive oxygen intermediates to eliminate invading microorganisms. The effectiveness of these and other microbicidal mechanisms is potentiated by the acidic pH created by proton-pumping V-ATPases on the phagosomal membrane. The degree of phagosomal acidification varies greatly between cell types, and can be affected by diseases like cystic fibrosis. The development of acidification is affected by the electrical potential that develops across the phagosomal membrane. We describe a non-invasive procedure to estimate phagosomal potential in intact cells, based on fluorescence resonance energy transfer. At steady state the phagosomal voltage averaged 27 mV (lumen positive) and was only partially dissipated by inhibition of the V-ATPase with concanamycin A. The comparatively small contribution of the potential to the protonmotive force suggests that in macrophages phagosomal proton pumping is not limited by the counter-ion permeability. Maintenance of luminal acidification was found to be essential for proper phagosomal maturation to phago-lysosomes. Dissipation of the proton gradient arrested maturation prior to acquisition of Rab7, despite the association of active Rab5 with the phagosomes and with endosomes. In fact, when the luminal acidification was abrogated, endosomes became enlarged and Rab5 was stimulated, leading to the accumulation of inordinately large amounts of phosphatidylinositol 3-phosphate. The phosphoinositide was ultimately delivered to the endosomal lumen, where it was sequestered and became unavailable to promote membrane fusion. We suggest that luminal acidification controls endosome and phagosome maturation, at least in part by signaling the timely termination of Rab5 activity.