The structural gene for the 49-kDa form of exoenzyme S (exoS) isolated from Pseudomonas aeruginosa 388 was expressed in both Escherichia coli and P. aeruginosa PA103. Expression of exoS in E. coli under the transcriptional regulation of the T7 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 49 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Expression of exoS in P. aeruginosa PA103 under the transcriptional regulation of the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS yielded a nitrilotriacetic acid-inducible extracellular protein with an apparent molecular mass of 49 kDa. Recombinant ExoS (rExoS) reacted with the anti-49-kDa form of exoenzyme S immunoglobulin G, existed as an aggregate as determined by gel filtration chromatography, and ADP-ribosylated soybean trypsin inhibitor at a specific activity that was similar (within twofold) to that of native exoenzyme S. Allelic exchange of exoS with a tetracycline gene cartridge yielded a strain of P. aeruginosa 388 that did not express detectable amounts of either ExoS in an immunoblot analysis using the anti-49-kDa form of exoenzyme S immunoglobulin G or ADP-ribosyltransferase activity under standard enzyme assay conditions. Expression of catalytically active rExoS in E. coli demonstrated that exoS was necessary and sufficient for the factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity of exoenzyme S. Expression of nitrilotriacetic acid-inducible rExoS in P. aeruginosa PA103 demonstrated that the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS encoded a functional exoenzyme S promoter. Expression analysis and allelic exchange experiments suggest that the 49- and 53-kDa forms of exoenzyme S are encoded by separate genes.