Abstract The eclipse of phage by F-piliated Escherichia coli has been shown to be an energy-requiring process with an apparent energy of activation of 10 kcal mole −1. The eclipse reaction was studied in glycerol- and glucose-grown cultures of E. coli in which ATP levels were varied by such metabolic poisons as anaerobiosis, sodium arsenate, and 2-deoxy- d-glucose. It was found that all the metabolic inhibitors studied caused the inhibition of phage eclipse when the cells were cultured in a glycerol medium. Moreover, there was a good correlation between the degree of inhibition of phage eclipse and the extent to which the ATP content of the cells was decreased. On the other hand, a correlation between inhibition of eclipse and decrease in cellular ATP levels was not found in the case of cells grown in glucose medium. In such cells, no inhibitor examined inhibited eclipsing by more than 14% although the ATP levels were greatly reduced in several instances. It was concluded that an energy donor other than ATP may be produced in glucose-grown cells, but not in glycerol-grown cells, and that this compound may be utilized to provide energy for the eclipse reaction. Additional evidence supports the possibility that this high-energy source may be membrane-associated.