We have examined its effect on the modulation of the intracellular accumulation and activity of daptomycin towards phagocytized S. aureus (ATCC 25923) in human THP-1 macrophages, in comparison with MDCK epithelial cells (wild type and MDCK-MDR1 overexpressing P-gp; the bulk of the protein was immunodetected at the surface of all three cell types). Daptomycin displayed concentration-dependent intracellular activity (Hill-equation pattern) in THP-1 and MDCK cells with (i) EC50 (50 % effective drug extracellular concentration; relative potency) and static concentrations at 9-10 x the MIC, and (ii) Emax (CFU decrease at infinite extracellular drug concentration) at 1.6-2 log compared to the post-phagocytosis inoculum. Verapamil (100 microM) and elacridar (GF 120918; 0.5 microM), two known inhibitors of P-gp, decreased daptomycin EC50 (about 3-fold) in THP-1 and MDCK cells without affecting Emax. Daptomycin EC50 was about 3-4-fold higher and accumulation in MDCK-MDR1 commensurately lower than in wild-type cells. In THP-1 macrophages, (i) verapamil and ATP depletion increased, and ouabain (an inducer of mdr-1 expression) decreased the accumulation of daptomycin in parallel with that of DiOC2 (a known substrate of P-gp); (ii) silencing mdr1 (the gene encoding P-gp) with duplex human mdr-1 siRNAs reduced the cell content in immunoreactive P-gp to 15-30 % of controls, and caused a 8-13-fold increase in daptomycin accumulation. We conclude that that daptomycin is subject to efflux from THP-1 macrophages and MDCK-cells by P-gp, which reduces its intracellular activity against phagocytized S. aureus.