The main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, K(ia) and K(a) (1.94+/-0.86 mM and 2.04+/-0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44+/-0.08 mM and 0.016+/-0.01 mM, respectively). Apparent dissociation constants of creatine, K(ib) and K(b) (2.12+/-0.21 mM 2.17+/-0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28+/-7 mM and 5+/-1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system.