31P-NMR has been used to study the increase of delta pH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P(i). Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0 degrees C, an increase of delta pH by approx. 0.5 pH unit was observed. However the increased delta pH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger delta pH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P(i) peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P(i) and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P(i) increase was stopped and the delta pH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.