Rotavirus causes enteritis in both man and animals. The mechanism by which this protein-enveloped virus enters the cell to initiate infection is not known. Many viruses depend on the acidification of endosomes for entry into the cell. We studied the importance of the acidification of endosomes for the initiation of two early phases of rotavirus infection, virus RNA and peptide synthesis. Monensin, nigericin, chloroquine and ammonium chloride at a variety of concentrations caused no specific inhibition of virus RNA or peptide synthesis. We conclude that the acidification of endosomes is not important for the entry of rotavirus into the cell.