Iron starvation interferes drastically with the multiplication and virulence of Salmonella typhi mutants defective in enterochelin synthesis or enterochelin transport. Growth of these mutants is inhibited in the presence of human sera and unsaturated transferrin and is restored by fully saturated transferrin. The mutants exhibit decreased ability to grow in HeLa cell monolayers and are attenuated in mice. These findings are consistent with the S. typhi enterochelin system playing a role in the pathogenesis of typhoid fever.