Macromolecules restore the morphological changes which occur upon isolation of mitochondria in normally used isolation media. It was shown that in the presence of dextrans the permeability of mitochondrial outer membrane for adenine nucleotides decreases which may have considerable implications for the transport of ADP into the mitochondria. In this study the effect of dextran on the apparent Michaelis constants of oxidative phosphorylation and mitochondrial creatine kinase (mi-CK) of rat heart mitochondria was investigated. Mitochondria were isolated either in normally used isolation media or in the additional presence of 15% dextran 20 in order to avoid changes in the oncotic conditions on the mitochondria during preparation and investigation. Except for an increased contamination with extramitochondrial ATPases the basic functional properties of these mitochondria were normal. With oxygraphic measurements it was found that Km(ADP) of oxidative phosphorylation increased from 16 +/- 4 microM ADP (without dextran) to 50 +/- 15 microM (15% dextran 20) and to 122 +/- 62 microM (25% dextran 20) irrespective of the mode of preparation of the mitochondria. Using spectrophotometric measurements the effect of dextran on the Km(ATP) of mi-CK was investigated in three systems (a) as soluble enzyme, (b) bound to mitoplasts, (c) and in intact rat heart mitochondria. The addition of 10% dextran had no effect on kinetic properties of solubilized mi-CK. In intact heart mitochondria, however, the addition of dextran caused an augmentation of Km(ATP) from 332 +/- 91 microM (control) to 525 +/- 150 microM ATP (10% dextran) and 641 +/- 160 microM ATP (30% dextran). In mitoplasts the effect of dextran disappeared (control, 230 +/- 19 microM ATP; 10% dextran, 238 +/- 28 microM ATP) indicating that the outer mitochondrial membrane is a prerequisite for the modulation of the transport of adenine nucleotides into the intermembrane space by macromolecules. To investigate the effects of viscosity of dextran solutions on the diffusion of adenine nucleotides across the outer membrane, dextrans with different molecular size (20, 40 70 and 500 kDa) were used. The viscosity of the 10% solutions drastically increased with the molecular size of the dextrans used, but the effects of different dextran solutions on the kinetic constants were the same. From these results it was concluded that neither the viscosity nor the molar concentration but the content of macromolecules (mass/vol.) correlates with restrictions of diffusion into the intermembrane space of mitochondria with intact outer membranes. Assuming that a dextran concentration of 15% mimicks the intracellular oncotic pressure on mitochondria in vivo, the apparent Km(ATP) of oxidative phosphorylation within the intact cell seems to be about 50 microM ADP which is somewhat higher than the cytoplasmic free ADP concentration as reported for the intact heart.