Protamine is a polycationic peptide found in the nuclei of sperm of different animal species. While it has long been known to have antimicrobial properties, its mode of action has remained elusive. We have investigated the mechanism of action of protamine and established that this peptide exerts its antibacterial effect without causing cell lysis or permeabilization of the cytoplasmic membrane. Respiring cells were more susceptible than nonrespiring cells, and loss of viability could be prevented by incubation at low pH or the addition of respiratory poisons. This indicates that protamine activity is influenced by the electrical membrane potential (delta psi): increased killing occurs at higher delta psi values. Protamine caused inhibition of proline uptake, rapid efflux of proline from preloaded cells, and a reduction in the cellular ATP content. Furthermore, protamine-treated cells first lost the ability to accumulate leucine and then could not carry out protein synthesis. Cumulatively, our data indicate that protamine disrupts energy transduction and nutrient uptake functions, and suggest that the cytoplasmic membrane is the target of protamine action.