Background: Huntington’s disease (HD) is associated with impaired energy metabolism in the brain. Creatine kinase (CK) catalyzes ATP-dependent phosphorylation of creatine (Cr) into phosphocreatine (PCr), thereby serving as readily available high-capacity spatial and temporal ATP buffering. Objective: Substantial evidence supports a specific role of the Cr/PCr system in neurodegenerative diseases. In the brain, the Cr/PCr ATP-buffering system is established by a concerted operation of the brain-specific cytosolic enzyme BB-CK and ubiquitous mitochondrial uMt-CK. It is not yet established whether the activity of these CK isoenzymes is impaired in HD. Methods: We measured PCr, Cr, ATP and ADP in brain extracts of 3 mouse models of HD – R6/2 mice, N171-82Q and HdhQ¹¹¹ mice – and the activity of CK in cytosolic and mitochondrial brain fractions from the same mice. Results: The PCr was significantly increased in mouse HD brain extracts as compared to nontransgenic littermates. We also found an approximately 27% decrease in CK activity in both cytosolic and mitochondrial fractions of R6/2 and N171-82Q mice, and an approximately 25% decrease in the mitochondria from HdhQ¹¹¹ mice. Moreover, uMt-CK and BB-CK activities were approximately 63% lower in HD human brain samples as compared to nondiseased controls. Conclusion: Our findings lend strong support to the role of impaired energy metabolism in HD, and point out the potential importance of impairment of the CK-catalyzed ATP-buffering system in the etiology of HD.