Creatine kinase activity is associated with different isozyme species. We have examined two of these: the cytoplasmic brain (B) isozyme that is expressed in many tissues and is reported to be induced by estrogen and the developmentally regulated cytoplasmic muscle (M) isozyme that is found predominantly in differentiated muscle tissue. Recently, we cloned and sequenced the cDNA for the M isoenzyme of rabbit creatine kinase. We now report the isolation of B-isozyme cDNAs and the deduced primary structure of the polypeptide. The translated cDNA nucleotide sequence was cross-checked by fast-atom bombardment/mass spectrometry of tryptic fragments from the protein. The sequence is exactly colinear with the rabbit M isozyme and the two isozymes have 80% nucleotide and amino acid sequence identity. There are blocks of 36 and 41 amino acids where the amino acid sequence is conserved exactly. The colinearity of the two sequences and the extent of their identity makes it unlikely that either isozyme has unique polypeptide domains that account for specialized functions. The rationale for the existence of these creatine kinase isozymes, with distinct biological features, evidently is at the level of regulation of individual isozyme expression.