We report the purification and proteolytic characterization of the 49-kDa form of exoenzyme S and the cloning of the structural gene for the 49-kDa form of exoenzyme S (exoS). The 49-kDa form of exoenzyme S was purified from SDS-polyacrylamide gels. Conditions were established that allowed efficient trypsin digestion of the 49-kDa form of exoenzyme S. Amino acid sequence determination of the amino terminus and tryptic peptides of the 49-kDa form of exoenzyme S allowed the generation of degenerate oligonucleotides, which were used to amplify DNA encoding an amino-terminal sequence and an internal sequence of the 49-kDa form of exoenzyme S. These DNA fragments were used to clone the entire structural gene for the 49-kDa form of exoenzyme S (exoS) from a cosmid library of Pseudomonas aeruginosa strain 388. The 49-kDa form of exoenzyme S (ExoS) is predicted to be a 453 amino acid protein. The predicted amino acid sequence indicates that ExoS is secreted from Pseudomonas without cleavage of an amino-terminal sequence. BESTFIT analysis identified three regions of alignment between ExoS and the active site of Escherichia coli heat-labile enterotoxin. One region of homology appears to be shared among several members of the family of bacterial ADP-ribosyltransferases.