Infection of MA-104 cells with the OSU strain of rotavirus induced an increase in Na+ and a decrease in K+ intracellular concentrations, starting at 4 h post-infection. These changes were not related to an inhibition of the Na+/K+ pump since ouabain-sensitive 86Rb uptake was augmented in rotavirus-infected cells compared to control cells, whereas the [3H]ouabain binding and Na+/K+ ATPase activity in the cell homogenate were unaffected. Furosemide-sensitive 86Rb uptake (Na+/K+/2Cl- cotransport) was not modified by the infection. Passive 86Rb efflux and 22Na influx were augmented in infected cells suggesting an increase in the plasma membrane permeability. The increase in intracellular Na+ concentration might be responsible for the observed stimulation of the Na+/K+ pump. This effect was dependent upon the synthesis of viral proteins because it was abolished by addition of cycloheximide up to 4 h post-infection. Prevention of the increase in intracellular Na+ by the use of low Na(+)-containing media did not modify the pattern of protein synthesis. This suggests that changes in intracellular Na+ and K+ concentrations were not related to shutoff of cellular protein synthesis. Alterations of ion contents in the rotavirus-infected enterocytes might impair intestinal absorptive capacity before the appearance of histopathological lesions.