The uptake of succinate by mitochondria and the rate of respiration in the presence of succinate plus rotenone have been analysed.1. I n conditions in which Vmax is the same, the apparent K , for succinate is lower when respiration is activated by ion uptake than it is in the active state. The highest value is obtained in the presence of 2,4dinitrophenol. 2. Succinate uptake is maximum during ion uptake, is lower in the active state and lowest in the uncoupled state.3. With increasing concentrations of uncoupler, a decrease in the intramitochondrial concentration of succinate is observed parallel to the decrease in respiration.4. The inhibition of respiration caused by gramicidin under certain conditions is accompanied by a decrease in the intramitochondrial concentration of succinate.5. Citrate and other substrate anions are shown to be competitive with succinate with respect to both the oxidation and the uptake of succinate.6. The inhibition of respiration by citrate is less pronounced in particles than in mitochondria. 7. It is concluded that the rate of respiration with succinate plus rotenone as substrate is controlled by succinate uptake.Phosphate and acetate are the best-known anions able to follow the movement of cations and accumulate within the mitochondria. Other anions which act in the same way have been reported by Azzi and Azzone [l], and Chance and Yoshioka [2]. Only recently has attention been drown to the behaviour of some mitochondrial substrates as anions [3-51. Important studies by Chappell and Haarhoff [6] showed that rat-liver mitochondria were permeable to some substrates, such as malate, succinate and citrate. Very recently in a note by Harris, van Dam, and Pressman [7] it was shown that the intramitochondrid concentration of succinate was decreased by high concentrations of uncouplers and increased in the presence of K+ and valinomycin. At the same time, similar results were also found by us and were the subject of a brief report 181. Furthermore, Rossi, Azzone, and Azzi 191 made the observation that succinate was bound to the mitochondria during Ca++ uptake and decreased the H+/Ca++ ratio from 2 to 1.I n this paper it is shown that succinate can accumulate within the mitochondria and that the rate of respiration supported by succinate plus rotenone is controlled by the accumulation of succinate, particularly when low concentrations of substrate are used.Non-Standard Abbreviations. DNP, 2,4-dinitrophenol; TMPD, tetramethyl-p-phenylenediamine.