A structural feature shared by many protein kinases is the requirement for phosphorylation of threonine or tyrosine in the so-called activation loop for full enzyme activity. Previous studies by several groups have indicated that the isotypes alpha, betaI, and betaII of protein kinase C (PKC) are synthesized as inactive precursors and require phosphorylation by a putative "PKC kinase" for permissive activation. Expression of PKCalpha in bacteria resulted in a nonfunctional enzyme, apparently due to lack of this kinase. The phosphorylation sites for the PKC kinase in the activation loop of PKCalpha and PKCbetaII could be identified as Thr497 and Thr500, respectively. We report here that PKCdelta, contrary to PKCalpha, can be expressed in bacteria in a functional form. The activity of the recombinant enzyme regarding substrate phosphorylation, autophosphorylation, and dependence on activation by 12-O-tetradecanoylphorbol-13-acetate as well as the Km values for two substrates are comparable to those of recombinant PKCdelta expressed in baculovirus-infected insect cells. By site-directed mutagenesis we were able to show that Thr505, corresponding to Thr497 and Thr500 of PKCalpha and PKCbetaII, respectively, is not essential for obtaining a catalytically competent conformation of PKCdelta. The mutant Ala505 can be activated and does not differ from the wild type regarding activity and several other features. Ser504 can not take over the role of Thr505 and is not prerequisite for the kinase to become activated, as proven by the unaffected enzyme activity of respective mutants (Ala504 and Ala504/Ala505). These results indicate that phosphorylation of Thr505 is not required for the formation of functional PKCdelta and that at least this PKC isoenzyme differs from the isotypes alpha, betaI, and betaII regarding the permissive activation by a PKC kinase.