1. 1. The interaction of butacaine with isolated rat liver mitochondria was investigated by monitoring the proton magnetic resonance spectrum of butacaine at 10° in 0.12 M KCl in D2O. 2. 2. Addition of mitochondria (1 mg mitochondrial protein to 0.3 μmoles butacaine) reduces by 50 per cent the intensity of each peak of the butacaine spectrum. The residual butacaine spectrum obtained in the presence of mitochondria exhibits no chemical shift. The resolution of the butacaine spectrum is partially lost in the presence of mitochondria, but is regained following separation of the mitochondria from the mixture. The entire butacaine molecule and not specific portions of it, appears to bind to mitochondria in such a manner that the bound butacaine contributes little to the spectrum of butacaine observed in the presence of mitochondria. 3. 3. Under conditions similar to those used to study the effect of mitochondria on the spectrum of butacaine, concentrations of the local anaesthetic within the range of 100-400 μM inhibit ADP translocation by about 40 per cent. 4. 4. The results are briefly discussed in relation to the mechanism by which butacaine inhibits adenine nucleotide translocation in mitochondria.