A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.