Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.