Creatine kinase (CK) is considered an important spatial and temporal energy buffer in the heart. It catalyzes the transfer of a phosphoryl group between creatine and ATP. Previous studies suggest that mice lacking cytosolic and mitochondrial CK exhibit cytoarchitectural modifications and changes in regulation of mitochondrial respiration in the heart. The aim of this study was to asses whether similar changes occurred in creatine-deficient mice lacking the creatine synthesizing enzyme guanidinoacetate methyltransferase (GAMT). More specifically, we wanted to characterize the modifications in cardiomyocyte mitochondrial organization, regulation of respiration and intracellular compartmentation associated with inhibition of the CK system by GAMT-deficiency. Mice received an i.p. injection of 250U heparin, and were anesthetized with ketamine/dexmedetomidine mixture (150 mg/kg and 0.5mg/kg, i.p.respectively). Cardiomyocytes were successfully isolated from 8 GAMT-/- (4 females and 4 males) and 9 WT mice (5 females and 4 males) of similar age (females WT 46.9 ± 4.9 weeks and GAMT-/- 45 ± 4.3 weeks; males WT 45.6 ± 1.5 weeks and GAMT-/- 45.9 ± 1.4 weeks). Three-dimensional mitochondrial organization at whole cell level was assessed by confocal microscopy.Kinetic measurements on permeabilized mouse cardiomyocytes included ADP and ATP-kinetics of respiration, the competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP-kinetics of endogenous pyruvate kinase and ATP-kinetics of ATPases. Using fluorescence microscopy, ADP-kinetics of respiration was confirmed on single permeabilized cardiomyocytes by recording autofluorescence response to changes in ADP. The experimental results were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of the morphologic and kinetic data, as well as derived model fits showed no difference between GAMT-deficient and wildtype littermates. Thus, we conclude that inhibition of the CK-system by GAMT-deficiency does not alter mitochondrial organization, regulation of respiration and intracellular compartmentation in relaxed cardiomyocytes. This raises questions on the importance of the CK system as a spatial energy buffer facilitating the communication between mitochondria and ATPases in cardiomyocytes.