In renal ischemia, tubular obstruction induced by swelling of epithelial cells might be an important mechanism for reduction of the glomerular filtration rate. We investigated ischemic cell swelling by examining volume regulation of A6 cells during metabolic inhibition (MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume were monitored by recording cell thickness (T_c). Intracellular pH (pH_c) measurements were performed with the pH-sensitive probe 5-chloromethyl-fluoresceine diacetate. T_c measurements showed that MI increases cell volume. Cell swelling during MI is proportional to the rate of Na⁺ transport and is not followed by a volume regulatory response. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. A transient acidification induced by a NH₄Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On the other hand, acute lowering of external pH to 5, simultaneously with the hypotonic shock, allowed the onset of RVD. However, this RVD was completely arrested ~10 min after the initiation of the hyposmotic challenge. The inhibition of RVD appears to be related to the pronounced acidification that occurred within this time period. In contrast, when external pH was lowered 20 min before the hyposmotic shock, RVD was absent. These data suggest that internal acidification inhibits cellular volume regulation in A6 cells. Therefore, the intracellular acidification associated with MI might at least partly account for the failure of volume regulation in swollen epithelial cells.