The factors contributing to the establishment of the steady state Golgi pH (pH G ) were studied in intact and permeabilized mammalian cells by fluorescence ratio imaging. Retrograde transport of the nontoxic B subunit of verotoxin 1 was used to deliver pH-sensitive probes to the Golgi complex. To evaluate whether counter-ion permeability limited the activity of the electrogenic V-ATPase, we determined the concentration of K ؉ in the lumen of the Golgi using a null point titration method. The [K ؉ ] inside the Golgi was found to be close to that of the cytosol, and increasing its permeability had no effect on pH G . Moreover, the capacity of the endogenous counter-ion permeability exceeded the rate of H ؉ pumping, implying that the potential across the Golgi membrane is negligible and has little influence on pH G . The V-ATPase does not reach thermodynamic equilibrium nor does it seem to be allosterically inactivated at the steady state pH G . In fact, active H ؉ pumping was detectable even below the resting pH G . A steady state pH was attained when the rate of pumping was matched by the passive backflux of H ؉ (equivalents) or "leak." The nature of this leak pathway was investigated in detail. Neither vesicular traffic nor H ؉ /cation antiporters or symporters were found to contribute to the net loss of H ؉ from the Golgi. Instead, the leak was sensitive to voltage changes and was inhibited by Zn 2؉ , resembling the H ؉ conductive pathway of the plasma membrane. We conclude that a balance between an endogenous leak, which includes a conductive component, and the H ؉ pump determines the pH at which the Golgi lumen attains a steady state.Stringent regulation of the internal pH of endomembrane compartments is required for their optimal function. In mitochondria, alkalinization of the matrix contributes to the generation of the transmembrane proton-motive force used to generate ATP (1). By contrast, luminal acidification is essential for the distribution and degradation of internalized ligands in the endocytic pathway (2-4), whereas it regulates post-translational modification and sorting of proteins along the secretory pathway (5-7). The pH varies in different subcompartments of the endocytic pathway, with acidification increasing progressively from the endocytic vesicles and early endosomes to late endosomes and ultimately lysosomes (8). Conversely, the pH of the secretory pathway becomes more acidic as the cargo travels toward the cell surface. Although the pH of the endoplasmic reticulum is thought to be near neutral (9, 10), acidification develops along the Golgi complex and is maximal at the transGolgi network (11-13). The development of such gradients appears to be important in targeting and retrieving components to and from individual subcompartments (6, 15). However, little is known about the determinants of pH in each subcompartment and particularly about the source of their differential acidification. In the case of the secretory pathway, this paucity of information is attributable, in part, to methodolo...