Ewing's family of tumors is characterized by a well described reciprocal translocation, t(11;22)(q24;q12), which produces a fusion protein (EWS/FLI-1) that transforms mouse fibroblasts. The EWS/FLI-1 fusion protein has been shown to act as a potent chimeric transcription factor. Overexpression of insulin-like growth factor-I receptor (IGF-IR) has been implicated in many tumor models as playing a role in cell growth and tumorigenesis. In addition, blockade of the IGF-IR inhibits the growth of Ewing's family of tumors cells. Therefore, we first studied whether the presence of the IGF-IR is required for transformation by the EWS/FLI-1 fusion protein. To perform this study, we used two previously described fibroblast cell lines, R- and W, derived from an IGF-IR knockout mouse and a wild-type littermate, respectively. Neither W nor R- cells without the fusion protein formed soft agar colonies. However, W clones expressing the fusion message (WF cells) formed soft agar colonies, whereas R- clones expressing the fusion message (R-F cells) did not form soft agar colonies. Because the IGF-IR is required for EWS/FLI-1 transformation, we chose to investigate whether altered signaling occurs from the IGF-IR when the EWS/FLI-1 fusion is present. WF cells demonstrated a greater degree of ligand-stimulated insulin receptor substrate-1 phosphorylation when compared with W cells, suggesting that expression of the EWS/FLI-1 fusion protein alters the IGF-IR signaling pathway.