Hybridization probes derived from the A and B subunit genes of the heat-labile enterotoxin (LT) of Escherichia coli were used to analyze DNA from Vibrio cholera strain 569B for cholera toxin gene sequences. Southern blot analysis indicated that the cholera toxin A and B subunit genes were each duplicated in the strain. One of the two toxin subunit gene pairs was cloned as a 5.1-kilobase DNA insert in plasmid pBR322. E. coli cells carrying the recombinant plasmid pJM17 were shown to produce cholera toxin, which was found to be largely cell associated. Protein chemical analysis indicated that the toxin was in its unnicked form and required additional proteolytic processing by trypsin to exhibit full toxicity in tissue culture. The alteration in E. coli of the secretion and proteolytic processing of cholera toxin parallels that previously observed for LT. An in vitro generated insertion mutation in the A subunit gene on pJM17 was shown to abolish production of the A chain but still allow production of the B chain. These observations, together with restriction mapping data, have demonstrated that the cholera toxin and LT genes are very similar in their genetic organization.