The binding of vesicular stomatitis virus (VSV) to Vero monkey cells was studied by using virus metabolically labeled with [35S]methionine. Under conditions where viral uptake did not occur (4 degrees C), apparent binding equilibrium was achieved within 12 h at a level representing 12% of the input virus. Two distinct forms of virus-cell interaction were found. At low concentrations of VSV, corresponding to multiplicities used for tissue culture studies, saturable binding was the major form of interaction. Saturation was complete at approximately 4,000 VSV virions per cell. At higher virus concentrations, nonsaturable binding prevailed. Trypsin treatment of Vero cells did not decrease the binding of VSV to the saturable binding sites. Internalization of VSV at 37 degrees C also displayed a saturable component which was directly comparable to that observed for binding. VSV binding to high-affinity, saturable sites on the plasma membrane may represent a receptor-mediated route of viral uptake.