In higher eukaryotes three different types of creatine kinases (CK) are expressed: the muscle-specific M-CK, the ubiquitous cytoplasmic B-CKs, and the mitochondrial Mi-CKs. They fulfill multiple tasks in cells with an intensive energy metabolism. Isolated chicken B-CK can be resolved by two-dimensional gel electrophoresis into a major acidic Ba-CK and a major basic Bb-CK protein species which are very likely produced from the unique chicken B-CK gene (Wirz, T., Hossle, J. P., Soldati, T., and Perriard, J.-C. (1989) Experientia (Basel) 45, 32 (abstr.]. However, close inspection of the gels indicates the presence of additional B-CK species. This additional heterogeneity is generated by two distinct post-transcriptional processes. Post-translational phosphorylation was shown to contribute to heterogeneity of both Ba- and Bb-CK isoproteins and appears to modulate their enzymatic activity (A. F. Q. Quest, H. M. Eppenberger, and T. Wallimann, manuscript in preparation). Alternative ribosomal initiation of Bb-CK synthesis at multiple sites was shown to occur in cell free systems as well as in vivo, resulting in proteins differing in the length of their amino termini. Using site-directed mutagenesis to "switch off" each of the first four methionine codons of a full length Bb-CK cDNA, we were able to correlate each protein product with one distinct translational start site. An additional protein species appears to be produced by initiation at a noncanonical start codon. We propose that a leucine codon may be used as a translational start site. Evidence is presented to support the role of these amino-terminal truncated subunits in the regulation of the enzyme.