The route of internalization of the pestivirus bovine viral diarrhea virus (BVDV) was studied by using different chemical and biophysical inhibitors of endocytosis. Expression of the dominant-negative mutant DynK44A of the GTPase dynamin in MDBK cells, as well as the treatment of the cells with chlorpromazine and β-methyl-cyclodextrin inhibited BVDV entry. BVDV infection was also abolished by potassium (K+) depletion, hyperosmolarity, and different inhibitors of endosomal acidification. We conclude that BVDV likely enters the cell by clathrin-dependent endocytosis and that acidification initiates fusion with the endosomal membrane. Further studies revealed that BVDV was unable to undergo "fusion from without" at low pH. The finding that low pH is not sufficient to force adsorbed BVDV into fusion with the plasma membrane is compatible with the remarkable resistance of pestiviruses to inactivation by low pH. The importance of the abundant intra- and intermolecular disulfide bonds in BVDV glycoproteins for virus stability was studied by the use of reducing agents. The combination of dithiothreitol and acidic pH led to partial inactivation of BVDV and allowed fusion from without at low efficiency. Evidence is provided here that acid-resistant BVDV is destabilized during endocytosis to become fusogenic at an endosomal acidic pH. We suggest that destabilization of the virion occurs by breakage of disulfide bonds in the glycoproteins by an unknown mechanism.