The deduced amino acid sequence of the cloned Salmonella enterotoxin gene (stn) was used to prepare anti-peptide antibodies. These antibodies were then employed to screen isolates of this enteric pathogen for the synthesis of protein enterotoxin (Stn). Cell lysates of all Salmonella isolates tested displayed a prominent immunoblot band of approximately 29 kDa, which was consistent with the size of the cloned stn gene product. Among other Gram-negative bacteria examined, isolates of Klebsiella, Enterobacter, and Citrobacter exhibited a similar-sized protein that reacted strongly with the Stn antibodies. Since the stn gene was located opposite the hydrogenase regulatory genes (hydHG) required for hydrogen metabolism in bacteria, our data suggested that only in Salmonella and some other members of the family Enterobacteriaceae had the DNA sequence evolved, presumably through point mutations, into an expressed gene product of similar size.