This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.