We used a probe specific for detecting the structural-gene sequences of Shiga toxin to analyze the genetic nature of toxin synthesis in mutant derivatives of Shigella dysenteriae type 1. A chlorate-resistant (chl) mutant (725-78) of S. dysenteriae type 1 strain 3818T, which had retained virulence but had lost production of high levels of cytotoxic activity associated with Shiga toxin synthesis, contained a complete deletion of the Shiga toxin structural-gene sequences. These structural-gene sequences were also absent in a derivative of S. dysenteriae type 1 that contained a substitution of Escherichia coli DNA in the trp region of the chromosome. Isolates of Shigella flexneri and Shigella sonnei also did not react with the probe. The low-level cytotoxic activities associated with the mutant S. dysenteriae type 1 strains or with the virulent S. flexneri and S. sonnei strains are neutralizable with antiserum to Shiga toxin; however, these cytotoxic activities are not determined by the genes encoding classic Shiga toxin.