The creatine kinase (CK) energy transport and buffering system supports cardiac function at times of high demand and is impaired in the failing heart. Mice deficient in muscle- and mitochondrial-CK (M/Mt-CK(-/-)) have previously been described, but exhibit an unexpectedly mild phenotype of compensated left ventricular (LV) hypertrophy. We hypothesised that heart failure would develop with age and performed echocardiography and LV haemodynamics at 1 year. Since all previous studies have utilised mice with a mixed genetic background, we backcrossed for >10 generations on to C57BL/6, and repeated the in vivo investigations. Male M/Mt-CK(-/-) mice on the mixed genetic background developed congestive heart failure as evidenced by significantly elevated end-diastolic pressure, impaired contractility, LV dilatation, hypertrophy and pulmonary congestion. Female mice were less severely affected, only showing trends for these parameters. After backcrossing, M/Mt-CK(-/-) mice had LV dysfunction consisting of impaired isovolumetric pressure changes and reduced contractile reserve, but did not develop congestive heart failure. Body weight was lower in knockout mice as a consequence of reduced total body fat. LV weight was not significantly elevated in relation to other internal organs and gene expression of LVH markers was normal, suggesting an absence of hypertrophy. In conclusion, the consequences of CK deficiency are highly dependent on genetic modifiers, gender and age. However, the observation that a primary defect in CK can, under the right conditions, result in heart failure suggests that impaired CK activity in the failing heart could contribute to disease progression.