CSK-Homologous Kinase (CHK) is a potential tumour suppressor in many types of cancer. It exerts its tumour suppressor action mainly by inhibiting the oncogenic Src-family tyrosine kinases (SFKs). CHK inhibits SFKs by, first, phosphorylating the conserved C-terminal regulatory tyrosine near the C-terminus of SFKs and by employing a non-catalytic inhibitory mechanism to inhibit SFKs. This latter mechanism involves directly binding of CHK to the kinase domain of SFK to form a stable complex, the binding alone is sufficient to inhibit SFK activity. This non-catalytic inhibitory mechanism is unique to CHK because the CSK (a homolog of CHK) can only inhibit SFKs by phosphorylating their C-terminal regulatory tyrosine. This project aims to decipher the structural basis of the non-catalytic inhibitory mechanism of CHK. In the crystal structure of a complex formed by CSK and the SFK member c-Src (c-Src:CSK complex), five basic residues in the kinase domain of CSK form ionic and hydrophobic interactions with the negatively charged and hydrophobic residues near the C-terminal tail c-Src. Since these five basic residues are conserved in CSK and CHK, it is likely that they also participate in binding of CHK to the SFK. Results of our studies indicate that all five residues including Arg-276, Arg-278 and Arg-280 in the αD helix and Arg-382 and Lys-387 in the αF-αG loop of the CHK kinase domain are determinants governing allosteric inhibition of SFKs by CHK, and Arg-382 and Lys-387 play significant role in the tight binding of CHK to SFKs. Since Arg-278 was recently discovered to be mutated in colon cancer, our results suggest that this mutation likely contributes to aberrant activation of the oncogenic SFKs and in turn colon cancer formation and progression by abolishing the non-catalytic inhibitory mechanism of CHK.