Fractional extraction and isozyme electrophoresis revealed the presence of small amounts (2.5% of total cellular activity) of mitochondrial creatine kinase (CK) in rabbit fast-twitch muscle. Chronic nerve stimulation resulted in a decrease of extramitochondrial MM-CK to 60% of its normal value but induced an approx. 4-fold increase in mitochondrial CK. This increase occurred in parallel with the rise in enzyme activities of terminal substrate oxidation.