The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue. This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide. But physiological targets of activated c-Src in mitosis have not yet been identified. Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts. p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo. p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides. Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.