A true protein kinase CKII (CKII) activity was characterized in liver mitochondria by its phosphorylating activity on the specific peptide substrate of CKII, the binding and elution profile of the enzyme on a phosphocellulose column and immunostaining of a 36 kDa polypeptide with antibodies against the alpha-subunit of human CKII. This CKII activity was located predominantly in the intermembrane space of quiescent mitochondria. A translocation of the enzyme to inner membrane of energized mitochondria occurred in the presence of spermine. Translocated CKII activity was tightly bound to inner membrane, and high salt concentrations were necessary to release the activity. The inner face of the inner membrane could constitute the in vivo localization of mitochondrial CKII since the potential substrates of the enzyme are 4 matrix proteins.