The cytosolic brain-type creatine kinase (BCK) isoform and the mitochondrial ubiquitous creatine kinase (UbCKmit) isoform are both important for the maintenance and distribution of cellular energy in neurons and astrocytes. Previously, we reported that mice deficient for BCK or UbCKmit each showed a surprisingly mild phenotype, probably due to reciprocal functional compensation by the remaining creatine kinase. This study shows that adult male mice lacking both creatine kinase isoforms (CK--/-- double knockout mice) have a reduced body weight, and demonstrate a severely impaired spatial learning in both a dry and a wet maze, lower nestbuilding activity and diminished acoustic startle reflex responses when compared to age-matched male wildtype mice with the same genetic background. In contrast, their visual and motor functions, exploration behaviour, prepulse inhibition and anxiety-related responses were not changed, suggesting no global deficit in sensorimotor function, hearing or motivation. Morphological analysis of CK--/-- double knockout brains revealed a reduction of approximately 7% in wet brain weight and hippocampal size, a approximately 15% smaller regio-inferior and relatively larger supra-pyramidal, and intra-infra-pyramidal mossy fiber areas. These results suggest that lack of both brain specific creatine kinase isoforms renders the synaptic circuitry in adult brain less efficient in coping with sensory or cognitive activity related challenges.