During the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that Na+/K+ ATPase activation is a consequence of Na+/H+ antiporter activation. Increased uptake of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione by infected cells around 2 h after infection suggested cytoplasmic alkalinization equivalent to pH 7.7 during the biosynthetic phase of viral replication. Consistent with the involvement of Na+/H+ antiporter activation in this phenomenon, it was found to be [Na+]-dependent and inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). However, the pH increase was not associated with an increase in amiloride-sensitive Na+ uptake by infected cells predicted by this mechanism. By contrast, the alkalinization could be abolished with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), implicating an anion-exchange mechanism, such as Cl-/HCO3- exchange, in this process. In addition to abolishing virus-induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular pH with nigericin in the incubation medium revealed that maximum viral replication required a pH of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the pH increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful pH increase in cells infected with a lytic virus.