The objective of this study was to examine the role of insulin-like growth factor II (IGF-II) in the pathogenesis of human rhabdomyosarcomas (RMS). We have demonstrated previously that RMS express high levels of IGF-II mRNA, secrete IGF-II peptide, and express both IGF-I and IGF-II receptors. Moreover, we showed that IGF-II functions as an autocrine growth and motility factor in RMS. Since IGF-II is expressed at high levels in fetal muscle cells and RMS are tumors thought to derive from skeletal myoblasts arrested along the normal myogenic pathway, autocrine production of IGF-II by RMS may be an etiological event in the development of this tumor. We have developed a model system which enabled us to study the effects of endogenous IGF-II overprotection in muscle myoblasts. Human cDNA for pre-prohormone IGF-II was transfected into mouse myoblasts in order to achieve high, constant expression of this growth factor, which is normally down-regulated at the end of the differentiation process. Expression of high IGF-II levels resulted in: (a) an increased proliferative rate; (b) impairment of the ability to differentiate into myoblasts; and (c) acquisition of the capability of anchorage-independent growth. No changes in the expression of IGF-I receptors were noted. We conclude that IGF-II overexpression in muscle myoblasts induces morphological and biological changes typical of the malignant phenotype and represents a fundamental event in the pathogenesis of RMS and possibly of other embryonal tumors.