Mutational alteration of the p16CDKN2a tumor suppressor gene has been frequently observed in a variety of human cell lines and tumor tissues. To assess whether alterations of the p16CDKN2a gene play an important role in the pathogenesis of Ewing's sarcoma, we examined the allelic deletion and point mutation of the gene in 27 primary tumors. In quantitative DNA/PCR analysis for three individual exons of the gene, none of the 27 specimens showed detectable reduction in the amplification levels compared to those of normal lymphocytes. To explore the presence of any subtle sequence changes within the protein coding region, we performed a comprehensive screening of sequence alteration in the three exons using DNA/PCR-SSCP analysis. However, no abnormal shift of SSCP bands indicative of sequence change was identified. In addition, no elevation of p16CDKN2a mRNA expression was observed in the RD-ES cell line following 5'-Aza-cytidine treatment, indicating that the promoter of the gene is not abnormally methylated in this cell line. Immunohistochemical study of the same tissue specimens for p16CDKN2a and pRB also revealed grade 2+ or 3+ nuclear staining of both proteins in most of the specimens we examined. Taken together, our results strongly suggest that the mutational inactivation of the p16CDKN2a gene might be a rare event, and thus not play a critical role in the pathogenesis of Ewing's sarcoma.