We have mapped a regulatory site mediating the hyperproduction of cholera toxin in mutants of Vibrio cholerae strain 569B. Mutations in this locus, called htx, result in the hypertoxinogenic phenotype, as measured by the ganglioside filter assay and immunoradial diffusion. Transposon-facilitated recombination was used to construct improved genetic donors in 569B parental and hypertoxinogenic mutant strains. Subsequent mapping by conjugation indicated that the htx locus was closely linked to the rif, str, and ilv loci of V. cholerae. Analysis of recombinants from these crosses suggested the following gene order: thy str htx rif ilv arg. The close genetic linkage of htx to rif (as high as 98%) resulted in a high comutation frequency of these two loci by nitrosoguanidine mutagenesis. Transfer of the htx mutant locus from a hypertoxinogenic donor to several unrelated Tox+ strains of V. cholerae caused a detectable elevation of toxin production in the recipients. These results suggest that toxin production in diverse strains of V. cholerae is controlled by a common regulatory mechanism in which the htx gene product plays a significant role.