Creatine kinase (CK; EC 2.7.3.2) catalytic activity in serum is widely measured in clinical chemistry practice and provides information for diagnosis and follow-up in many pathological conditions affecting heart, muscle, and brain. Depending on the organ involved, the predominant CK isoenzyme in serum varies. However, routine methods measure total CK catalytic activity, and standardized methods for doing so have been recommended by the International Federation of Clinical Chemistry and by several national scientific societies. Many commercial kits for those methods are now available. With use of a reference material for CK, commercial reagents can be compared with standardized methods, improving confidence in the results. Here we present a reference preparation of CK consisting of the BB isoenzyme purified from human placentae. We describe the procedure of purification and the properties of the lyophilized preparation of CK-BB, which has been certified by the Community Bureau of Reference of the Commission of the European Communities under the designation CRM 299. The preparation can be used to calibrate assays of the catalytic activity of CK-MM and CK-MB, as well as CK-BB.