Previous studies have shown that channel formation in the cytoplasmic membrane of Escherichia coli by colicin A and phage T5 leads to an efflux of cytoplasmic potassium and to a membrane depolarization. Here we show that upon opening of these channels, the intracellular ATP concentration is decreased to 10% of its original value in < 5 min. ATP is not found in the external medium, but is hydrolyzed in the cytoplasm into ADP and AMP. The rate of ATP hydrolysis depends on the number of channels and on their activity. ATP hydrolysis takes place if the F1F0-ATPase is absent or inhibited. Depolarization of the inner membrane is not the main cause of ATP hydrolysis. Opening of the phage and colicin channels also leads to an efflux of inorganic phosphate. Conditions that prevent the efflux of phosphate (i.e. depolarization of the cells and high external phosphate concentration) prevent ATP hydrolysis. We propose that ATP is hydrolyzed as a consequence of a shift in the ATP equilibrium due to the efflux of phosphate through the channels. The consequences for the cells of this ATP depletion are discussed.