A WKA rat fetus-derived fibroblast cell line WFB showed strict nontransformant phenotypes in vitro such as anchorage dependency of cell growth in soft agar, contact inhibition, and serum dependency on the monolayer cell culture. Transfection of 6.6-kilobase EJras oncogene into WFB resulted in the acquisition of tumorigenicity in vitro and in vivo. The cell surface antigen that is moderately or highly expressed on these WFB transformants, designated as W14 and W31, was analyzed using monoclonal antibody 109 that was produced after the immunization of BALB/c mice with W31. Moab 109 recognized a glycoprotein with a molecular weight of 36,000 composed of a single polypeptide chain with 5.4 isoelectric point value. This antigen was highly expressed on WFB EJras and polyoma middle T-DNA transformants, but was undetectable or at the best only faintly recognized on WFB parental cells, transfectants of WFB with c-myc, and normal thymus, liver and kidney of WKA adult rats. It was also clearly expressed on the EJras transformants of Fisher rat fetus-derived 3Y1 fibroblast, but very faintly on parental 3Y1. Furthermore, this antigen was detected on some rat T-lymphoma and gliosarcoma lines. However, it was undetectable on EJras transformants on NRK-49F rat kidney cells and NIH3T3 and BALB3T3 mouse cells. In addition, this antigen appeared on the cell surface of concanavalin A-activated WKA rat lymphocytes and WKA rat on the 16th day of embryo but not on the 8th. These results suggested that the cell surface antigen detected by Moab 109 was clearly unrelated to the ras oncogene product p21 that was highly expressed on EJras-transformants of WFB or 3Y1 cells. Furthermore, it was shown that W14 and W31 cells but not parental WFB cells were susceptible to rat splenic NK cells that were induced by poly(I-C) treatment. Pretreatment of these W14 or W31 cells with Moab 109 could block the NK cell activity against W14 and W31. These data suggest that this antigen may act as one of the NK target structures, and plays an important role as a tumor antigen on the host tumor surveillance, since the antigen was expressed (a) on the cell surface after the cell transformation or enhanced DNA synthesis of some particular cells, and (b) in the W31 tumor developing progressively in the syngeneic rats.