The effects of the bactericidal protein colicin K have been compared on a normal strain of E.coli and a derivative, uncA, that is defective in Ca2+, Mg2+-activated ATPase activity. The syntheses of DNA, RNA, and proteins, which are completely arrested by colicin K in the normal E. coli cells, are only reduced by 30 to 50% in the ATPase-deficient cells. Intracellular ATP levels, which are reduced in the normal cells, are substantially increased by colicin K in the uncA mutant. The active transport of glutamine, which is coupled to phosphate bond energy, is drastically inhibited in both strains as is the transport of proline, a transport system that is coupled to an energized membrane state. These results provide the basis for the proposal that the primary action of colicin K is to de-energize the cytoplasmic membrane systems that are essential for a variety of active transport processes in E. coli. In cells having ATPase activity, ATP levels are diminished by the action of ATPase attempting to re-energize the membrane. This reduction in ATP levels in turn leads to an arrest of macromolecular biosyntheses. These secondary effects of colicin K on ATP levels and on synthetic processes are not present in uncA cells because they lack ATPase activity.