We demonstrated free flow electrophoresis (FFE) of charged cells under microgravity, where gravitational effects are almost eliminated. Separation of a mixture of three bacterial strains (mutants of Salmonella typhimurium LT2) by FFE was conducted on NASA Space Shuttle flight STS-47 (September 1992). The experiment was designed to differentiate three strains having different lipopolysaccharide core structures in the cell membrane. The results were compared to those of ground experiments, in order to examine whether or not FFE in a weightless environment provides distinct advantages. Smooth strain SL1027 and rough strain SL3749 migrated to two separated fractions. The quality (viability) and the yields of the separated samples were sufficient to show the advantage of microgravity. Another rough strain, SL1102, exhibited unexpected electrophoretic behavior, which prevented the complete resolution of the three strains. All the strains were recovered as viable cells after 8 days of flight. The present study suggests that electrophoretic separation of bacterial cells is much more effective under microgravity conditions with relatively good resolution in comparison with the ground operation.