Background and objective: One of the main causes of bacterial resistance to antimicrobials is multidrug resistance induced by the increased efficiency of the efflux pumps. In this study we analyzed how the conditions of assay affect the efflux of indicator substrates ethidium (Et+) and tetraphenylphosphonium (TPP+) in Salmonella enterica ser. Typhimurium cells. Impact of the outer membrane permeability barrier, composition and temperature of the medium on accumulation of the indicator compounds also was analyzed.Materials and methods: The fluorescence of Et+ and Nile Red was measured using 96-well plates and a plate reader. In parallel to traditional studies of fluorescence we applied a constructed selective electrode to follow the accumulation of Et+ in S. enterica cells. Simultaneously with monitoring of Et+ concentration in the cell incubation medium, electrochemical measurements of TPP+ accumulation were performed. Furthermore, Et+ and TPP+ were used within the same sample as agents competing for the interaction with the efflux pumps. An inhibitor phenylalanyl-arginyl-β-naphtylamide (PAβN) was applied to evaluate the input of RND-family pumps in the total efflux of these indicator compounds.Results: S. enterica cells with the intact outer membrane (OM) bound very low amounts of Et+ or TPP+. Cells with the permeabilized OM accumulate considerably higher amounts of the indicator compounds at pH 8.0, but only Et+ was considerably accumulated at pH 6.5. At conditions of electrochemical monitoring accumulation of Et+ by the permeabilized cells at 37 °C was considerably faster than at 23 °C, but at the higher temperature most of the cellaccumulated Et+ was extruded back to the medium. The fluorescence of Et+ in suspension of cells incubated in 400 mmol/L Tris buffer was about twice higher compared to 100 mmol/L one. The inhibitory action of TPP+ on Et+ efflux was evident only in 400 mmol/L Tris although PAβN effectively increased Et+ fluorescence at both buffer concentrations.Conclusions: Results of our experiments indicate that ionic strength of the incubation medium influence the selectivity, the medium temperature and the assay conditions impact the kinetics of efflux. The lower accumulated amount and the weaker fluorescence of Et+ registered in slightly acidic medium indicate that ΔΨ plays a role in the accumulation of this indicator cation. The bound amount of Et+ to the de-energized or permeabilized cells considerably varies depending on the conditions and methods of de-energization or permeabilization of cells. Tris/EDTA permeabilization of the cells does not inhibit the efflux.