We applied Western blotting, using an antibody against the carboxy terminal of the FLI1 protein, for detection of the 68-kDa EWS/FLI1 fusion protein in cultured Ewing's sarcoma cells and in four surgical biopsies of Ewing's sarcoma. Of six different human cell lines, the 68-kDa fusion protein was identified only in Ewing's sarcoma cells carrying the t(11;22)(q24;q12) translocation. The four samples from Ewing's sarcoma patients were also found to contain the 68-kDa fusion protein. The lowest detection level for total protein loaded on the gel was 0.3 microg. When whole Ewing's sarcoma cells were used for Western blotting without prior protein extraction, the lowest detection level was 1,300 cells. It will be possible to use Western blotting for detection of the EWS/FLI1 fusion protein in the diagnosis of Ewing's sarcoma in surgical biopsy specimens, and possibly also in fine-needle aspirates. As the method is not dependent on the quality of mRNA in the sample and involves no risk of contamination, it will be a powerful complement to the reverse-transcription polymerase chain reaction (RT-PCR).