Exoenzyme S is an ADP-ribosyltransferase enzyme distinct from exotoxin A that is synthesized and secreted by Pseudomonas aeruginosa. Yields of exoenzyme S are variable and depend on strain and growth conditions. Since certain medium additives are required for exoenzyme S production, its regulation may be influenced by environmental stimuli. In this study, we have cloned a region that complements the exoenzyme S-deficient phenotype of strain 388 exs1::Tn1, a chromosomal Tn1 insertional mutation. A large clone (28 kb) was shown to restore both synthesis and secretory functions to the mutant strain. Subcloning and Tn501 mutagenesis experiments localized the region required for exoenzyme S synthesis to a 3.2-kb fragment. Nucleotide sequence analysis demonstrated several open reading frames. Comparison of the N-terminal amino acid sequence of purified exoenzyme S with predicted amino acid sequences of all open reading frames indicated that the structural gene was not encoded within the sequenced region. Homology studies suggested that the region encoded three regulatory genes, exsC, exsB, and exsA. ExsA was homologous to the AraC family of transcriptional activator proteins, with extensive homology being found with one member of this family, VirF of Yersinia enterocolitica. VirF and ExsA both contain carboxy-terminal domains with the helix-turn-helix motif of DNA-binding proteins. The ExsA gene product appeared to be required for induction of exoenzyme S synthesis above a low basal level. Expression of ExsA was demonstrated by cloning the region under the control of the T7 promoter. Gene replacement experiments suggested that the expression of ExsC affects the final yield of exoenzyme S.