1  Department of Molecular and Cell Biology and 2  Department of Physics, University of California, Berkeley, California 94720-3200 Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pH G ) of HeLa cells was 6.4, while pH G of mutant ( F508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7-7.0. Comparison of genetically matched F508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H + vacuolar (V)-ATPase countered by a H + leak, which was unaffected by CFTR. To estimate Golgi proton permeability ( P H + ), we modeled transient changes in pH G induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi P H +   = 7.5 × 10 4 cm/s when pH G  = 6.5, and surprisingly, P H + decreased as pH G decreased. enhanced yellow fluorescent protein; ultraviolet enhanced green fluorescent protein; trachea; organelle pH; proton permeability; cystic fibrosis