Comparison of cholera toxin (CT) production directed by different gene constructs and S1 nuclease mapping revealed the presence of a ctxB-specific promoter within the ctxA coding sequence. Initiation of transcription in this region occurred in wild-type El Tor and classical biotype choleragenic vibrios. We propose that transcription from the ctxB-specific promoter and a stronger ribosomal binding site on the ctxB mRNA synergistically contribute to achieve the correct (5B:1A) subunit stoichiometry. Plasmid pB, a CT promoterless vector expressing only CTB, was used to detect promoter activity by restoration of A-subunit synthesis. Promoter activity expressed in vitro and in vivo was detected upstream of the zonula occludens toxin gene, suggesting that this factor could be produced in vivo to contribute to fluid accumulation. No promoter activity was detected in vitro and in vivo upstream from the accessory cholera enterotoxin gene.