Hippocalcin (Hpca) is a Ca2+-binding protein that is expressed in neurons and contributes to neuronal plasticity. We purified a 48 kDa Hpca-associated protein from rat brain and identified it to be the creatine kinase B (CKB) subunit, which constitutes brain-type creatine kinase (BB-CK). Hpca specifically bound to CKB in a Ca2+-dependent manner, but not to the muscle-type creatine kinase M subunit. The N-terminal region of Hpca was required for binding to CKB. Hpca mediated Ca2+-dependent partial translocation of CKB (approximately 10-15% of total creatine kinase activity) to membranes. N-myristoylation of Hpca was critical for membrane translocation, but not for binding to CKB. In cultured hippocampal neurons, ionomycin treatment led to colocalization of Hpca and CKB adjacent to the plasma membrane. These results indicate that Hpca associates with BB-CK and that together they translocate to membrane compartments in a Ca2+-dependent manner. (C) 2012 Elsevier Inc. All rights reserved.