During the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that N a t / K + ATPase activation is a consequence of N a + / 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 p H 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 CI-/HCO,-exchange, in this process. In addition to abolishing virus-induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular p H with nigericin in the incubation medium revealed that maximum viral replication required a p H of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the p H increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful p H increase in cells infected with a lytic virus.