Background: Creatine kinase (CK) and hexokinase (HK) play a key role in myocardial energy homeostasis. We aimed to determine CK and HK expression and enzyme activity in the left (LV) and right (RV) ventricles of rats adapted for 3 weeks to normobaric hypoxia (10 % O2) either continuously (CNH) or intermittently with 1-h or 16-h normoxic episode per day. Methods: The Real-Time RT-PCR, Western blot, and enzyme-coupled assays were used. In addition, the effect of CNH on the HK co-localization with mitochondria, which can inhibit apoptosis, was assessed using immunofluorescence techniques. Results: CK and HK activities increased in the LV during all hypoxic adaptations, which was consistent with elevated protein levels of mitochondrial mtCKs, cytosolic CKB, HK1, and HK2 isoforms. Enzyme activities also increased in the hypoxic RV, but only CKB protein was elevated. No effect of CNH on HK1 or HK2 co-localization with mitochondria was observed. Conclusion: Up-regulation of mtCKs and HK isoforms may stimulate the respiratory chain and help to maintain energy homeostasis of chronically hypoxic myocardium and prevent oxidative stress. In this way, CK and HK enzymes can possibly participate in the establishment of ischemia-resistant phenotype of chronically hypoxic hearts.