Autophosphorylation in the activation loop is a common mechanism regulating the activities of protein tyrosine kinases (PTKs). PTKs in the Csk family, Csk and Chk, are rare exceptions for lacking Tyr residues in this loop. We probed the function of this loop in Csk by extensive site-specific mutagenesis and kinetic studies using physiological and artificial substrates. These studies led to several surprising conclusions. First, specific residues in Csk activation loop had little discernable functions in phosphorylation of its physiological substrate Src, as Ala scanning and loop replacement mutations decreased Csk activity toward Src less than 40%. Second, some activation loop mutants, such as a single residue deletion or replacing all residues with Gly, exhibited 1-2% of wt activity toward artificial substrates, but significantly higher activity toward Src. Third, introduction of a thrombin cleavage site to the activation also resulted in loss of 98% of wt activity for polyE4Y and loss of 95% of wt activity toward Src, but digestion with thrombin to cut the activation loop, resulted in full recovery of wt activity toward both substrates. This suggested that the catalytic machinery is fully functional without the activation loop, implying an inhibitory role by the activation loop as a regulatory structure. Fourth, Arg313, although universally conserved in protein kinases, and essential for the activity of other PTKs so far tested, is not important for Csk activity. These findings provide new perspectives for understanding autophosphorylation as a regulatory mechanism, and imply key differences in Csk recognition of artificial and physiological substrates.