Malignant melanoma in the fish Xiphophorus is induced by overexpression of the Xmrk-oncogene, encoding a subclass I receptor tyrosine kinase. The mutationally activated Xmrk protein triggers constitutive mitogenic signalling in fish melanoma cells. In recent studies we showed that in melanoma cells phosphatidylinositol (PtdIns) 3-kinase, as well as p59fyn, has elevated levels of kinase activity. Both bind directly to different phosphotyrosine residues in the Xmrk receptor C-terminus through their SH2 domains. To analyse the mechanism of regulation of these Xmrk-associated kinases in melanoma we characterized the protein-protein interactions between PtdIns 3-kinase, p59fyn and the Xmrk receptor in detail. A ternary complex in which the p85 subunit of PtdIns 3-kinase is associated with p59fyn as well as with Xmrk was identified. Contrary to complexes described for other receptors, the adaptor protein p120Cbl was not involved in these interactions. Thus, we describe here a new mechanism of activation of PtdIns 3-kinase by a receptor of the epidermal growth factor receptor family in which p59fyn acts as an adaptor as well as an activator of PtdIns 3-kinase. Activation of PtdIns 3-kinase activity by fyn was also found in vivo. The fact that this was only detectable in highly transformed Xmrk overexpressing melanomas but not in benign lesions points to the essential role of the Xmrk receptor in this mechanism of regulation.