The values reported in the literature for the extramitochondrial ATP/ADP ratio in resting rat-liver mitochondria (State 4) vary widely. The conditions required for an accurate determination of this parameter were therefore investigated. In experiments with rat-liver mitochondria incubated under State-4 conditions, it was found that the extramitochondrial ATP/ADP ratio, as calculated from the values measured in neutralised perchloric acid extracts, was lower than that estimated from the concentrations of creatine and creatine phosphate, using the metabolite indicator method. The discrepancy is due to hydrolysis of ATP occurring in the presence of perchloric acid. Conditions are described for minimising ATP hydrolysis in the presence of perchloric acid, and include the use of low concentrations of perchloric acid, short times of exposure to the acid before neutralisation, low temperatures and the presence of excess EDTA. Under these conditions, the values obtained for the extramitochondrial ATP/ADP ratio agreed with those calculated by the metabolite indicator method, provided ratios do not exceed the value of 100. In cases where the extramitochondrial ATP/ADP does exceed 100, phenol/chloroform/isoamyl alcohol must be used to quench the reactions, as described by Slater et al. (Slater, E.C., Rosing, J. and Mol, A. (1973) Biochim. Biophys. Acta 292, 534-553). With this method, the extramitochondrial ATP/ADP ratio was found to have a value of more than 1000 in rat-liver mitochondria incubated with succinate + rotenone in the resting state (pH 7.0; T = 37 degrees C), in agreement with Slater et al.