In cardiac muscle, the creatine kinase (CK) system temporally buffers ADP and ATP concentrations near sites of ATP production and consumption. It has also been suggested to be an important spatial buffer facilitating the transport of ADP and ATP. This was corroborated by a study of CK knockout mice, where cardiac fibers exhibited changes in mitochondrial organization and functional compartmentation – potentially to diminish diffusion distances between mitochondria and ATPases. However, a recent study on cardiomyocytes from creatine-deficient guanidinoacetate methyltransferase (GAMT) knockout mice showed no effect on mitochondrial organization and compartmentation. It has been suggested that in GAMT-/- mice, accumulated guanidinoacetate can be used as a substrate instead of creatine. Therefore, we studied the same parameters in L-arginine:glycine amidinotransferase (AGAT) mice, which are also creatine-deficient, but do not accumulate guanidinoacetate. 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). Three-dimensional mitochondrial organization at whole cell level was assessed by confocal microscopy. Kinetic measurements on permeabilized cardiomyocytes included the affinity of oxidative phosphorylation to exogenous ADP and ATP, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP-kinetics of endogenous pyruvate kinase and ATP-kinetics of ATPases. Tibial length of AGAT-/- mice was similar to that of AGAT+/+ mice, but body weight was significantly lower. Our results show surprisingly little difference between cardiomyocytes of AGAT-/- and AGAT+/+ mice. We did find that the protein content of AGAT-/- cardiomyocytes was approximately two times lower than that of AGAT+/+ cardiomyocytes. However, visual inspection of mitochondrial organization suggests no essential difference. Also, when respiration is related to the number of cardiomyocytes, there is no difference in the kinetics of respiration and ATPases. Thus, our data so far suggest no differences in cardiomyocyte mitochondrial organization and cellular compartmentation between AGAT-/- and AGAT+/+ mice. We conclude that the lack of compensatory changes in mitochondrial organization and cellular compartmentation in AGAT as well as GAMT mice raise questions regarding the importance of the CK system as a spatial buffer.