The EWS-ETS rearrangements, and their respective fusion gene products, are specifically associated with histopathologically Ewing family tumors (EFT). These translocations are implicated in generating malignant transformation of EFT, but the presence of additional genetic alterations must be considered in the pathogenesis of such tumors. We analyzed 26 samples (biopsies and/or nude mice xenotransplants) collected from 19 patients with an EFT to determine whether molecular and cytogenetic alterations of the G(1)/S checkpoint genes are implicated in the pathogenesis of EFT. We found inactivating p53 mutations in three (16%) cases, which correlated with a loss of p21(WAF1/Cip1) expression and with a monosomy of chromosome 17 in two cases. Homozygous deletion of the p16(INK4A)/p14(ARF) gene was detected in four (21%) cases, three with codeletion of the p15(INK4B) gene and with chromosome 9 abnormalities. In all of these cases, expression of the implicated genes was absent. Hypermethylation of the p16(INK4A) and p15(INK4B) genes was detected in two (10%) and three (16%) cases, respectively, and was correlated with a low level of gene expression. Neither cyclin D1, nor MDM2 and CDK4 amplification was observed. Kaplan-Meier analysis showed that patients with tumors carrying homozygous deletion of the 9p21 locus, or point mutations of the p53 gene, had poorer outcomes than those without these molecular alterations (p = 0.005). In conclusion, 58% (11 of 19) of the analyzed patients showed genetic or epigenetic alterations in either the 9p21 locus or p53 tumor suppressor genes, defining a subgroup of patients with poor clinical outcome. This fact points to an important role of the G(1)/S cell cycle checkpoint dysregulation in the pathogenesis of EFT.