The activity of the c-Src tyrosine kinase is regulated through intramolecular interactions between the catalytic and SH2/SH3 domains. However, the exact mechanism by which this occurs remains obscure. In the crystal structure of c-Src, the peptide that links the SH2 and catalytic domain (SH2-CD linker) is sandwiched between the latter and the SH3 domain. A residue in the linker, Leu 255, inserts its side chain into a deep hydrophobic pocket present on the surface of the catalytic domain. To investigate the possible regulatory role of this prominent interaction, we mutated Leu 255 to different hydrophobic residues. We found that the length and 'bulkiness' of the side chain had a profound influence on c-Src regulation. Src-L255V was highly active but showed reduced SH3 accessibility in vitro as well as an altered localization in vivo when compared to other deregulated forms of Src. Our analyses lead us to suggest that the Leu 255-pocket interaction is a critical component of the intramolecular inhibition mechanism of Src family kinases.