We have cloned a positive regulatory gene ( toxR ) from Vibrio cholerae that controls cholera toxin transcription. This was done by first constructing a genetic fusion consisting of the lacZ gene fused to the promoter of the cholera toxin operon ctxAB . This operon fusion was used to screen a V. cholerae genomic library for genes that could activate the ctx promoter in Escherichia coli. This method allowed the identification of a gene, toxR , that increases ctx expression by more than 100-fold. Complementation analysis indicated that certain hypotoxinogenic mutants of V. cholerae 569B probably have mutations in the toxR gene. Southern blot analysis suggests that all V. cholerae, including nontoxinogenic strains, have the toxR gene. Moreover, nontoxinogenic strains not only lack the structural genes for cholera toxin but also sequences associated with the larger 7-kilobase ctx genetic element.