Sam68 is a 68 kDa protein that associates with and is phosphorylated by the c-Src kinase at mitosis. It contains a KH domain implicated in RNA binding and several proline-rich motifs that resemble known SH3 binding sites. The SH3 domains of c-Src, phosphatidylinositol 3-OH kinase, phospholipase C-gamma and Grb2 protein (containing two SH3 domains), but not other SH3 domains tested, were capable of binding Sam68 in vitro. Synthetic peptides corresponding to the proline motifs of Sam68 inhibited with different efficiencies the binding of SH3 domains to Sam68 suggesting that the proline motifs of Sam68 function as specific SH3 domain binding sites. Mutation of Sam68 SH3 binding sites further indicated that the SRC SH3 domain mediates binding of Src to unphosphorylated Sam68. Phosphorylation of Sam68 by Src kinase was inhibited when the Src SH3 binding site of Sam68 was mutated or when corresponding peptides were added to in vitro kinase reactions indicating that binding of the Src SH3 domain to a specific site near the amino-terminus of Sam68 (including residues 38 - 45: PPLPHRSR) facilitates phosphorylation of Sam68 by the Src kinase domain. Sam68-based proline peptides had no effect on the phosphorylation of another in vitro substrate of Src, enolase. These results suggest that Src effectively mounts Sam68 through its SH3 domain, possibly as a mechanism to position the kinase domain close to substrate tyrosine residues in the carboxyl-half of the protein.