Background: Mammalian organelles of the secretory pathway are of differing pH. The pH values form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral, the Golgi is mildly acidic and the secretory granules are more acidic still (pH 5). The mechanisms that regulate pH in these organelles are still unknown.

Results: Using a novel method, we tested whether differences in H⁺ ‘leak’ and/or counterion conductances contributed to the pH difference between two secretory pathway organelles. A pH-sensitive, membrane-permeable fluorescein–biotin was targeted to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa cells. In live, intact cells, ER pH (pH_ER) was 7.2 ± 0.2 and Golgi pH (pH_G) was 6.4 ± 0.3 and was dissipated by bafilomycin. Buffer capacities of the cytosol, ER and Golgi were all similar (6–10 mM/pH). ER membranes had an apparent H⁺ permeability three times greater than that of Golgi membranes. Removal of either K⁺ or Cl⁻ did not affect ER and Golgi H⁺ leak rates, or steady-state pH_G and pH_ER.

Conclusions: The Golgi is more acidic than the ER because it has an active H⁺ pump and fewer or smaller H⁺ leaks. Neither buffer capacity nor counterion permeabilities were key determinants of pH_G, pH_ER or ER/Golgi H⁺ leak rates.