Salmonella typhimurium is a facultative intracellular pathogen capable of surviving within phagocytic cells of the reticuloendothelial system. To identify the genes important for intracellular survival, 9516 independent Tn10 insertional mutations were isolated in a virulent strain of S. typhimurium. By using an in vitro assay for survival within macrophages, 83 Tn10 mutants have been identified that have a diminished capacity for intracellular survival (designated MS or macrophage survival mutants). All of the MS mutants are less virulent than the parent strain in vivo, demonstrating that, for Salmonella, survival within the macrophage is essential for virulence. Thirty-seven of the MS mutants have been characterized as to their phenotype, including several mutations that confer sensitivity to specific microbiocidal mechanisms of the macrophage.