The two most recent hypotheses about the histogenesis of Ewing's Sarcoma (ES) are that it has a mesenchymal or neuroectodermal origin. Immunologic markers specific to these two tissue origins were tested on cryostat sections from three primary tumors carrying the chromosomal translocation t (11; 22)(q24; q12). Cell lines established in vitro from two of these three primary tumors were also analyzed. Using antibodies directed against neural components (neurone-specific-enolase [NSE], HNK-1, and neurofilament triplet proteins [NFTP]), positive reactions were observed in cells from two primary tumors and their corresponding cell lines. Results of electron microscopic examination of the primary tumors were compatible with the diagnosis of ES. When using antibodies directed against mesenchymal cell surface antigens (common leucocytes, Leu M1, Leu M2, and Leu M3), the weak positive reactions observed in the three primary tumors were attributed to lymphoid infiltrates within tumor cells. Six additional ES cell lines carrying the translocation t (11; 22) were also analyzed by immunocytochemical and flow cytometry methods using antibodies directed against mesenchymal and neural components. Positive reactions were observed in all seven cell lines tested using antibodies directed against NSE, HNK-1, and 200 KD subunit of the NFTP, whereas negative reactions were obtained with Leu M2 antibody. These results are consistent with a neuroectodermal origin of ES cells.