Phosphorylation of the region containing Thr-494, Thr-495 and Thr-497, present in the catalytic domain of protein kinase C alpha (PKC alpha), is a preliminary event necessary for subsequent PKC activation [Cazaubon and Parker (1993) J. Biol. Chem. 268, 17559-17563]. To define the essential residues in this region, various combinations of alanine substitutions for threonine residues 494, 495 and 497 have been tested. These mutations yielded expressed polypeptides of 76 and 80 kDa in ratios that vary from 100% 80 kDa (wild-type kinase, active) to 100% 76 kDa (AAA mutant, inactive) with the hierarchy being wild-type PKC alpha (TTT), ATT, AAT, TTA, ATA, TAA, AAA (the nomenclature indicates the location of alanine residues substituted for Thr-494, Thr-495 and Thr-497 respectively). Only the mutants retaining Thr-497 displayed kinase activity in vitro. The results overall indicate that Thr-497 plays the dominant role in the regulation of PKC alpha activity but that in the wild-type protein, Thr-495 may also be important. Consistent with the need for phosphorylation in this region, an intrinsically active PKC alpha could be produced in bacteria by exchanging Thr-495 for a glutamic acid residue.