Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane's Na(+) and Ca(2+) pumps. The formation of the Na(+)- and Ca(2+)-dependent phosphointermediates of both types of pumps (E(Na)-P and E(Ca)-P) that conventionally can be labeled with trace amounts of [gamma-(3)P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [gamma-(3)P]ATP in the pool. Running the pumps forward with either Na(+) or Ca(2+) removes pool ATP and allows the normal formation of labeled E(Na)-P or E(Ca)-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10-15 minutes. We observed that when unlabeled "caged ATP" is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E(Na)-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na(+) (without Mg(2+)) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of (45)Ca by the Ca(2+) pump, analogous to the Na(+) pump uptake of (22)Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na(+) and Ca(2+) pumps.