1. Creatine kinase activities in cytosol and mitochondrial fractions of rat submandibular gland were separated clearly by DEAE cellulose column chromatography and by electrophoresis on cellulose acetate strips. 2. Creatine kinase in cytosol fraction was brain type. 3. A portion of mitochondrial creatine kinase was tightly bound to mitochondria, and was solubilized with Triton X-100. 4. Mitochondrial creatine kinase was separated into 2 portions by DEAE cellulose column chromatography, but not by electrophoresis and gel filtration. 5. Kinetic analysis indicated mitochondrial creatine kinase was directed for the formation of creatine phosphate.