The brain-type (BB) isoenzyme of creatine kinase (CK) has recently been shown to be estrogen-regulated in human breast cancer cells in vitro. In this study we have correlated the presence of CK-BB, evaluated by immunoperoxidase procedures, with the estrogen receptor (ER) content in 35 primary breast cancers. Of 35 tumors examined, 66% revealed moderate to strong CK-BB immunoreactivity. Staining was exclusively located in the cytoplasm of neoplastic cells. A positive relationship was observed between CK-BB positivity and ER content with ER-rich tumors (greater than 50 fmoles/mg protein) showing a more intense immunoreactivity than ER-poor ones. The results indicate that CK-BB is estrogen-regulated in human breast cancer and suggest that evaluation of this enzyme may be of potential value for the detection of hormone responsive tumors.