(1) (1)The effects of nupercaine and other local anaesthetics on anion transporters in isolated rat heart mitochondria were investigated at temperatures of 25° or 30°. The concentrations of nupercaine which inhibited by 50 per cent the rates of pyruvate-stimulated oxygen utilisation in (a) the presence and (b) the absence of α-cyano-3-hydroxycinnamate, (c) ADP-stimulated oxygen utilisation in the presence of atractyloside, (d) succinate-stimulated oxygen utilisation in the presence of phenylsuccinate (e) phosphate-induced swelling were (a) 0.02, (b) 0.4, (c) 0.3, (d) 1.1 and (e) 0.6 mM, respectively. (2) (2)Low concentrations of nupercaine (10-50 μM) increased by about 10 per cent the rates of ADP- and succinate-stimulated oxygen utilisation measured at 30° in the presence of atractyloside and phenylsuccinate, respectively. (3) (3)Inhibition by nupercaine of pyruvate-stimulated oxygen utilisation in the presence of α-cyano-3-hydroxycinnamate was found to be non-competitive with respect to pyruvate. (4) (4)Butacaine and tetracaine inhibited pyruvate-stimulated oxygen utilisation, but were less effective than nupercaine. Procaine and xylocaine had little effect. (5) (5)[2-14C]Pyruvate uptake was inhibited by nupercaine with 50% inhibition given by 0.2 and 0.6 mM nupercaine at 25° and 4° respectively. (6) (6)The concentrations of nupercaine which inhibited pyruvate-stimulated oxygen utilisation in the presence of 2-cyano-3-hydroxycinnamate had no effect on the rate of mitochondrial swelling or the acceptor control ratio of the mitochondria. (7) (7)It is concluded that the pyruvate transporter is markedly more susceptible to the inhibitory effects of nupercaine than are the adenine nucleotide. dicarboxylate or phosphate ion transporters.