Purification of human mitochondrial creatine kinase has been difficult and procedures that were highly successful in purifying canine enzyme failed for human mitochondrial creatine kinase. In the present study, we employed ultracentrifugation to remove the lipid, urea to prevent aggregation, followed by a final step of chromatofocusing which yielded a preparation of human mitochondrial creatine kinase with a specific enzyme activity of greater than 400 IU/mg. Biochemical and immunological characterization showed the preparation to be highly pure and free of even trace amounts of other creatine kinase isoenzymes. Antiserum specific for mitochondrial creatine kinase was developed which exhibited no cross-reactivity to cytosolic creatine kinase and mitochondrial creatine kinase did not cross-react with antiserum to the cytosolic forms. Marked differences were noted, both biochemically and immunologically, between mitochondrial creatine kinase and the cytosolic forms. Human mitochondrial creatine kinase was shown to have a molecular weight of around 82,000 and to be composed of two subunits of equal molecular weights around 41,000. Aggregates of mitochondrial creatine kinase were observed with molecular weights of around 200,000 in the absence of urea or if isolated from material after having undergone proteolysis. Isolation from fresh material or in the presence of urea inhibited aggregate formation for both canine and human mitochondrial creatine kinase. Despite claims of several investigators that mitochondrial creatine kinase exhibits two to three forms with varying molecular weights, our data indicate a single enzyme form made up of a subunit with a molecular weight of 41,000 and the high molecular weight aggregates appear to be induced artifacts. A radioimmunoassay was developed for human mitochondrial creatine kinase which, with appropriate modifications, should detect mitochondrial creatine kinase in human plasma.