Six syngeneic rat cell lines transformed with isolated or cloned left end fragments of adenovirus type 12 (Ad12) DNA were used in a study of the Ad12-specific cell surface antigen. Cells transformed with EcoRI-C, SalI-C, and HindIII-G fragments of Ad12 DNA fragment-transformed express the E1a and a part of or a complete E1b early regions. Two AccI-H DNA fragment-transformed cell lines contain and express only the E1a region. All these cells contain nuclear Ad12 antigen. Cytotoxic antibodies raised against syngeneic EcoRI-C DNA fragment-transformed cells kill EcoRI-C, SalI-C and HindIII-G fragment-transformed cells, but fail to kill cells transformed with AccI-H DNA fragment. Immunofluorescence analysis shows that such antibodies, which stain the surface of cells expressing the E1b region, do not stain the surface of AccI-H DNA fragment-transformed cells. Cells transformed with AccI-H fragment are also not killed by secondary cytolytic T cells, raised and effective against cells transformed with EcoRI-C, SalI-C, and HindIII-G DNA fragments. Cells transformed with AccI-H fragment do not elicit cytolytic T cells against any of the studied cell lines. The only Ad12-specific product shared by all cell lines killed in cytolytic assays which is absent from AccI-H fragment-transformed cells is the E1b 18K protein. Since it has also been shown in other studies that this protein is associated with the cellular membrane, the simplest interpretation of these data is that the Ad12-specific cell surface antigen in transformed rat cells is a product of the left end of the E1b region.