A rabbit antiserum (A2) directed against the detergent-solubilized fraction of the simian virus 40-transformed mouse embryo fibroblast cell line VLM detects common antigens in primary cell cultures from BALB/c mouse embryos and in transformed cell lines from various species. Positively reacting cell cultures show a set of polypeptides with molecular weight species p86, p74, p68, p46, p42, p40, and p35. As tested by Western blotting procedures, all immunoprecipitated proteins carry immunologically reactive determinants. By analysis with two-dimensional gel electrophoresis, all precipitated polypeptides show charge heterogeneities. Concerning the two major members of the protein set, p40 consists of at least four subspecies with isoelectric points in the range of pH 6.2-6.8, whereas p35 is composed of two subspecies focusing between pH 6.4 and pH 7.2. By comparison of the two-dimensional patterns of p35 of various transformed cell lines, a basic (pH 6.6-7.2) and an acidic (6.4-6.6) charge type of p35 could be observed. Comparative analyses of primary cell cultures from 12-16-day mouse embryos show the immunoprecipitated set of polypeptides only in the 16-day embryo cell cultures. After six further propagations, these cells express the immunoreactive proteins as strongly as the primary cell cultures. In embryonic cell cultures of day 14 of gestation the expression of this set of antigens is induced only when cells are propagated at least six times. Under identical conditions these proteins could not be induced in cell cultures of 18-day-old mouse embryos. None of the polypeptides could be immunoprecipitated from primary mouse kidney cell cultures of 12-day-old mice even when the cultures were propagated at least 15 times. This set of polypeptides is also present in simian virus 40-transformed cells of hamster, rat, monkey, and human origin. These findings suggest that in simian virus 40-transformed mouse cells, in addition to p53, the synthesis of other embryonic antigens is reactivated. The presence of the described set of polypeptides in polyoma virus-transformed cells of rat and mouse origin and in cell lines derived from malignant human tumors might indicate common functions in metabolic patterns of transformed cells.