During the last decade many reports on the isolation from plant tissues of particulate fractions containing many respiratory enzymes, have been published (5). These fractions proved to be very suitable for a detailed study of respiratory metabolism, the more so because plant mitochondria which after addition of substrate and cofactors gave ?2 uptake coupled with phosphorylating processes, could be isolated. The effect of 2,4-dinitrophenol upon the respiration rate of potato tuber tissue (15) strongly suggests that under normal conditions respiratory activity and oxidative phosphorylation are coupled, and regulated by the availability of phosphate acceptor (ADP). Therefore, also after isolation, the mitochondria should have a substrate oxidation which is coupled with phosphorylation, and the rate of which is controlled by ADP. Hackett et al. (8) were the first who described the ability of potato tuber mitochondria to carry out oxidative phosphorylation. In their experiments using the Warburg respirometer and using succinate as the substrate they found P/O ratios of around 1.3, but they did not obtain evidence for a respiratory control by ADP. The best method available for investigating respiratory control in mitochondria is the oxygen electrode method (9) which allows short-term experiments. Bonner and Voss (2) using this highly sensitive method were the first who succeeded in demonstrating a respiratory control by ADP in isolated plant mitochondria. Later Wiskich and Bonner (17) were able to prepare mitochondrial fractions from potato tuber tissue, which not only showed accelerated 02 uptake upon addition of ADP, but also a decreased rate of substrate oxidation when the ADP had been consumed. They foundl respiratory control ratios (R.C. ratios) of around 2.0. Although Hackett et al. (8) and Wiskich and Bonner (17) made great progress in isolating mitochondria from potato tuber tissue, their preparations had some properties which suggested that the condition of the particles might not have been very good. Wiskich and Bonner could already eliminate the marked increase of the respiration upon addition of cytochrome c, as described by Hackett et al. (8), but their preparations exhibited a considerable induction period for the ADP stimulation of the succinate oxidation. Such an induction period could be avoided by preincubation of the mitochondria with ATP. Now it is known that ATP has an effect on the maintenance of contraction in mitochondria (7, 13). So the ATP induction phenomenon may be an indication that the mitochondria require a recovery, induced by ATP, from changes occurring during the isolation procedure, before they can react to addition of ADP. Therefore, the conditions for the isolation of mitochondria from potato tuber tissue have been reinvestigated in order to obtain mitochondria in a better condition than described up until now.