Among primitive neuroectodermal tumors, Ewing tumors are characterized by a gene rearrangement recombining the 5'-EWS gene portion with one of two ETS-related proto-oncogenes, FLI-1 or ERG, in roughly 90 and 10% of cases, respectively. We attempted to antagonize EWS/FLI-1 function in Ewing tumor cell lines. As a control, a cell line derived from another small round cell tumor of neuroectodermal origin, neuroblastoma, was used. This cell line was found to express almost identical patterns of ETS proteins except for EWS/FLI-1 and a novel, ELF-related gene product. Stable expression of antisense EWS/FLI-1 cDNA resulted in decreased endogenous EWS/FLI-1 RNA levels and growth reduction of ET cells but not of neuroblastoma cells. DNA-binding FLI-1 derivatives localizing to the nucleus in which the 5'-EWS regulatory domain was replaced by bacterial beta-galactosidase dominantly modulated transcriptional transactivation from a degenerate ETS-binding motif. Transient transfection of these dominant-negative recombinants resulted in a significant decrease in the relative number of mitoses in four Ewing tumor cell lines tested but not in the neuroblastoma cell line. The presented evidence for modulation of tumor cell proliferation by EWS/FLI-1 antagonists suggests a causal role for EWS/FLI-1-mediated gene activation in the malignant transformation of the enigmatic Ewing tumor-precursor cell.