A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.