Tr-kit is a truncated form of the tyrosine kinase receptor c-kit expressed in the haploid phase of spermatogenesis. Upon microinjection, tr-kit triggers metaphase-to-anaphase transition in mouse eggs by the sequential activation of Fyn and PLCgamma1. Here, we show that tr-kit promotes the interaction of several tyrosine-phosphorylated proteins with the SH3 domain of PLCgamma1. Western blot analysis indicates that one of these proteins is Sam68, an RNA-binding protein that is known to interact with and be phosphorylated by Src-like kinases in mitosis. tr-kit promotes the association of Sam68 with PLCgamma1 and Fyn in a multimolecular complex, as demonstrated by co-immunoprecipitation of the phosphorylated forms of these proteins using antibodies directed to anyone of the partners of the complex. Expression of tr-kit potentiates the interaction of endogenous Sam68 also with the SH3 domain of Fyn. Furthermore, the subcellular localization of Sam68 is affected by tr-kit through activation of Fyn in live cells. Lastly, we show that interaction with the SH3 domain of Fyn triggers the release of Sam68 from bound RNA. Thus, our data suggest that tr-kit promotes the formation of a multimolecular complex composed of Fyn, PLCgamma1 and Sam68, which allows phosphorylation of PLCgamma1 by Fyn, and may modulate RNA metabolism.