In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb* was identified by chromatofocussing in the presence of 8 M urea (Quest et al., ). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bb1-3), initially identified in immunoprecipitated, [35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [32P]-gamma-ATP, Bb1-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb* is identical to Bb1-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the Km values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb* two kinetically different enzyme species were detected (Km values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb*, were homogeneous in this respect (Km for CP = 1.6 mM). Phosphorylation of Bb to yield Bb* is concluded to reduce the Km of B-CK dimers for CP by about 50%. This Km shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.