Muscle-type creatine kinase (MM-CK) is a member of an isoenzyme family with key functions in cellular energetics. It has become a matter of debate whether the enzyme is autophosphorylated, as reported earlier [Hemmer, Furter-Graves, Frank, Wallimann and Furter (1995) Biochim. Biophys. Acta 1251, 81-90], or exclusively nucleotidylated. In the present paper, we demonstrate unambiguously that CK is indeed autophosphorylated. However, this autophosphorylation is not solely responsible for the observed microheterogeneity of MM-CK on two-dimensional isoelectric focusing gels. Using phosphoamino-acid analysis of (32)P-labelled CK isoforms, phosphothreonine (P-Thr) residues were identified as the only product of autophosphorylation for all CK isoenzymes. The phosphorylated residues in chicken MM-CK were allocated to a region in the vicinity of the active site, where five putative phosphorylation sites were identified. Site-directed threonine-valine-replacement mutants reveal that autophosphorylation is not specific for one particular residue but occurs at all examined threonine residues. The enzyme kinetic parameters indicate that the autophosphorylation of CK exerts a modulatory effect on substrate binding and the equilibrium constant, rather than on the catalytic mechanism itself.