We describe a selection method for mutants altered in the generation and regulation of transmembrane ion flux in Halobacterium halobium. The method is based on experimental control of ion fluxes by a combination of light, ionophore, and external pH to generate an imbalance in the cells' proton circulation through their membranes. The steady-state proton circulation is increased by the introduction of a small inward proton leak with a protonophore. The cells are then illuminated to excite halorhodopsin, which hyperpolarizes the membrane and drives protons into the cells. As a result, wild-type cells suffer cytoplasmic acidification, which causes a dramatic loss of motility and suppresses their growth. These properties can be used to select for mutants that escape cytoplasmic acidification because either they lack halorhodopsin function or they have a greater capacity to eject protons during the illumination. In a popular selected by this method, 97% of the individual cells were demonstrably altered in ion flux properties. Cells were selected with alterations in the halobacterial rhodopsin, specifically with deficiencies in membrane potential generation by halorhodopsin and with increased cellular proton ejection by bacteriorhodopsin. We describe properties of one of the halorhodopsin-deficient strains, Flx37.