The gene encoding cytotoxin (ctx) was cloned from Pseudomonas aeruginosa 158 and the nucleotide sequence was determined. The structural gene of ctx encodes the procytotoxin of 286 amino acid residues with a molecular mass of 31,681 Daltons. Procytotoxin was activated by removal of 20 amino acid residues from the C terminus with trypsin. The cloned ctx gene was not expressed in either an Escherichia coli strain or a cytotoxin non-producing strain of P. aeruginosa. An expression system for the ctx gene was constructed by placing the structural gene of ctx downstream of tac promoter on a broad host-range vector plasmid.