A WKA rat fetus fibroblast (WFB) was transfected by several oncogenes including EJras (activated H-ras), polyoma middle T (PyMT), v-src, c-myc, and adenovirus type 12 E1A-E1B. We analyzed the expression of the transformation-associated cell surface antigens on WFB by developing monoclonal antibodies. One of the WFB transformation-associated cell surface antigens, recognized by monoclonal antibody 067, was constitutively expressed only on two (W31 and W70) of ten WFB-EJras-transformed clones. This antigen could not be detected on parental WFB cells as well as ten WFB-PyMT transformant clones. Furthermore, it was not expressed on several clones of partially transformed WFB-v-src and WFB-adeno E1 transfectants or nontransformed WFB-c-myc transfectants. Monoclonal antibody 067 could form an immunoprecipitate with an approximate molecular weight of 67,000 which was composed of a single polypeptide chain. It was also shown that the expression of this antigen could be enhanced by cyclic AMP or cholera toxin treatment of W31; treated cells also showed a phenotypic reversion to the nonmalignant growth characteristics of the parental WFB. Moreover, the expression of this antigen could be induced on the WFB-EJras transformants such as W14, which do not constitutively express this antigen, by treatment of these agents. Furthermore, the expression of antigen was enhanced by heat and superoxide treatment on W31. These data suggest that the monoclonal antibody 067-defined molecule is a novel transformation-associated cell surface antigen that could be induced by heat shock or other physiological stress.