The catalytic subunit of cAMP dependent protein kinase (cAPK) carries two stable autophosphorylated residues. One of them, Thr197, resides in the so-called protein kinase activation segment, and needs to be phosphorylated for full activity and protein kinase inhibitor binding of the enzyme. While wild-type recombinant mammalian C-subunit, expressed in E. coli, can fully autoactivate itself by phosphorylation at Thr197, many active site mutants lack this autophosphorylation activity, so that the primary effects of the mutations become obscured. Two active site mutants of bovine C-subunit, defective in protein kinase inhibitor peptide binding, were activated by wild-type enzyme in vivo, but could not be activated in vitro, demonstrating intermolecular and presumably cotranslational autophosphorylation. The results may delineate strategies for the expression and mutagenesis of other protein kinases with requirements for activation segment phosphorylation.