The sources of energy and energy coupling reactions of the active transport systems for isoleucine and proline in Escherichia coli cells were studied. It was concluded that the isoleucine transport system utilizes ATP molecule as an energy source and that the initial rate of isoleucine uptake is directly correlated with the cellular concentration of ATP. Active uptake of proline, on the other hand, is generated by an “energized” membrane state of the cells and does not depend on the cellular concentration of ATP. Results suggested that a coupling factor of oxidative phosphorylation generates the “energized” membrane state utilizing cellular ATP in the absence of respiration. All studies were carried out using Escherichia coil strain NR-70 which lacks membrane-bound, Mg2+ -requiring adenosine triphosphatase [EC 3.6. 1.3]. The NaN3-sensitive, Mg2+ adenosine triphosphatase and the respiratory and oxidative phosphorylating activities of the mutant were extensively analyzed. It was concluded that the NaN3 Mg2+-requiring adenosine triphosphatase functions as a coupling factor of oxidative phosphorylation and that the mutant is defective in this enzyme activity.