c-Src is a tightly regulated non-receptor tyrosine kinase. We describe the C-terminus of c-Src as a ligand for a PDZ (postsynaptic density 95, PSD-95; discs large, Dlg; zonula occludens-1, ZO-1) domain. The C-terminal residue Leu of c-Src is essential for binding to a PDZ domain. Mutation of this residue does not affect the intrinsic kinase activity in vitro, but interferes with c-Src regulation in cells. As a candidate PDZ protein, we analysed AF-6, a junctional adhesion protein. The AF-6 PDZ domain restricts the number of c-Src substrates, whereas knockdown of AF-6 has the opposite effect. Binding of c-Src to the AF-6 PDZ domain interferes with phosphorylation of c-Src at Tyr527 by the C-terminal kinase, and reduces c-Src autophosphorylation at Tyr416, resulting in a moderately activated c-Src kinase. Unphosphorylated Tyr527 allows binding of c-Src to AF-6. This can be overcome by overexpression of CSK or strong activation of c-Src. c-Src is recruited by AF-6 to cell-cell contact sites, suggesting that c-Src is regulated by a PDZ protein in special cellular locations. We identified a novel type of c-Src regulation by interaction with a PDZ protein.