Calcium ions (Ca 2+) play a pivotal role in eukaryote cell signaling and regulate many physiological functions. Although a similar role for Ca 2+ in prokaryotes has been difficult to demonstrate, there is increasing evidence for Ca 2+ as a cell regulator in bacteria. The purpose of this study was to investigate Ca 2+ signaling and the effect of Ca 2+ on the Staphylococcus aureus multidrug resistant efflux pump LmrS. We hypothesized that antibiotics act by increasing Ca 2+ concentrations, which in turn enhance the efflux activity of LmrS. These Ca 2+ transients were measured by luminometry in response to various antibiotics by using the photoprotein aequorin reconstituted within live bacterial cells. Efflux associated with LmrS was measured by the increase in fluorescence due to the loss of ethidium bromide (EtBr) from both S. aureus cells and from E. coli cells in which the lmrs gene of S. aureus was expressed. We found that addition of antibiotics to cells generated unique cytosolic Ca 2+ transients and that addition of CaCl 2 to cells enhanced EtBr efflux whereas addition of Ca 2+ chelators or efflux pump inhibitors significantly decreased EtBr efflux from cells. We conclude that antibiotics induce a Ca 2+ mediated response through transients in cytosolic Ca 2+ , which then stimulates LmrS efflux pump.