A new nondefective hybrid virus has been plaque-isolated from the Ad.2-SV40 hybrid population. This virus replicates efficiently with one-hit kinetics in both human embryonic kidney and African green monkey kidney cells, induces an SV40 specific antigen which is detectable by immunofluorescence and complement-fixation using sera from SV40 tumor-bearing hamsters, and produces SV40-specific RNA detectable by DNA-RNA hybridization. The SV40-specific antigen induced by this virus is heat-stable, sensitive to inhibitors of DNA synthesis, serologically different from SV40 T and viral antigens, and is an unrecognized SV40 antigen.