Creatine (Cr), the substrate of the creatine kinase (CK) isoenzymes, was shown to be neuroprotective in several models of neurodegeneration, including amyotrophic lateral sclerosis (ALS). In order to investigate the mechanism of this beneficial effect, we determined CK activities and mitochondrial respiration rates in tissues from G93A transgenic mice, which overexpress a mutant form of human superoxide dismutase associated with familial ALS (FALS). While respiration rates of mitochondria from G93A transgenic or wild-type control mice isolated from spinal cord showed no difference, a significant and dramatic loss of CK activity could be detected in these tissues. In homogenates from spinal cord of G93A transgenic mice, CK activity decreased to 49% and in mitochondrial fractions to 67% compared to CK activities in wild-type control mice. Feeding the G93A transgenic mice with 2% Cr, the same tissues showed no statistically significant increase of CK activity compared to regular fed G93A transgenic mice. Experiments with isolated mitochondria, however, showed that Cr and adenosine triphosphate (ATP) protected mitochondrial CK activity against peroxynitrite-induced inactivation, which may play a role in tissue damage in neurodegeneration. Our data provide evidence for oxidative damage to the CK system in ALS, which may contribute to impaired energy metabolism and neurodegeneration.