The main efflux pump of Salmonella enterica serotype Enteritidis, which obtains its energy for the extrusion of noxious agents from the proton-motive force, was studied with the aid of an ethidium bromide (EtBr) semi-automated method under conditions that define the role of metabolic energy, ions and pH in the extrusion of the universal substrate EtBr. The results obtained in this study indicate that in minimal medium containing sodium at pH 5 efflux of EtBr is independent of glucose, whereas at pH 8 metabolic energy is an absolute requirement for the maintenance of efflux. In deionised water at pH 5.5, metabolic energy is required for the maintenance of efflux. The inhibitory effect of the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) on efflux is shown to be minimised by low pH, and at high pH by metabolic energy. Similarly, thioridazine, an inhibitor of metabolic enzymes, inhibits efflux of EtBr only at pH 8 and the degree of inhibition is lessened by the presence of metabolic energy.