Our work focused on pH changes in cytosol (pH_i) and mitochondria (pH_m) in metabolically inhibited (MI) MDCK cells, a cell line of distal tubular origin. MI was applied for 1 h, by inhibiting both cellular glycolysis (with 2-deoxyglucose) and oxidative phosphorylation (with NaCN). Changes in pH_i and pH_m were detected by confocal microscopy on confluent living cells grown on glass coverslips. Cell monolayers were doubly labelled with carboxy SNARF-1 (a ratiometric pH sensitive probe) and MitoTracker Green (a specific mitochondrial probe, used as a "mask" to discriminate between the cytosolic and matrix SNARF signal). The dual emission ratio of SNARF (680 nm/590 nm) was monitored as a measure for pH. MI resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of pH_m as compared to pH_i. During the first 20 min of MI, pH_i decreased from 7.4 ± 0.1 to 7.1 ± 0.1 (N=5) and exhibited a slight recovery for the subsequent 40 min of treatment. The pH_m decreased from 7.7 ± 0.1 to 7.0 ± 0.1 during the first 20 min of MI and continuously decreased down to 6.8 ± 0.1 (N=7) in the next 40 min of MI. Cells showed a delayed recovery of the pH_i and pH_m upon removal of metabolic inhibitors. This delay might act as a protective mechanism, possibly preventing the initiation of apoptosis. As supported by confocal and electron microscopy images, MI induced reversible mitochondrial swelling and network disruption. Morphological changes during MI correlate with changes in mitochondrial Na⁺ mediated by mitochondrial Na⁺/Ca²⁺ exchanger working in a normal/reverse mode, as we presented previously (AJP 286: F784-794, 2004; JASN 16: 3490-7, 2005).