The nature of the simian virus 40 (SV40) receptor on the surfaces of Vero C1008 cells was investigated by a virus binding assay. The optimum pH for SV40 binding to cell surfaces was found to be at 6.5; however, there was little difference in SV40 binding in the range between pH 4.5 and 7.3. The treatment of cell surfaces with several proteases or with an enzyme specific for O-linked carbohydrates significantly reduced virus binding, suggesting that the receptor for SV40 contains protein and O-linked carbohydrates. Treatment of cell monolayers with octyl glucoside removed virus-binding activity from cell surfaces. Recovery of virus-binding activity by octyl glucoside-treated cells took 2.5 h and was inhibited by cycloheximide or tunicamycin. Four polypeptides with molecular weights of 90,000, 58,000, 54,000, and 30,000 were immunoprecipitated from virus-protein complexes derived from octyl glucoside extract solutions and therefore may be components of the SV40 receptor. Competition experiments between SV40 and polyomavirus revealed that these two viruses do not share the same receptor on Vero C1008 cells.