We have previously confirmed the estrogen-induced protein of rat uterus to be creatine kinase B (CKB), and demonstrated a 1.7-kilobase pair fragment encompassing the promoter and adjoining 5'-flank to be capable of conferring estrogen responsiveness in HeLa cells. In this study we find an element at -550, aGGTCAgaaCACCCt, with limited similarity to the estrogen response element consensus, to be involved in conferring estrogen responsiveness on the CKB promoter. This element can bind estrogen receptor (ER) and is flanked by two GC boxes, which we find capable of binding bacterially expressed Sp1. Additional responsiveness is found closely associated with the CKB promoter at high levels of cotransfected ER construct. No potential response element was identified in this region, but we find the ER DNA-binding domain to be required.