The full elastolytic phenotype of Pseudomonas aeruginosa requires lasB, the structural gene for elastase, its transcriptional activator lasR, and lasA. The lasB gene was insertionally inactivated with the omega fragment and this mutated gene introduced into the P. aeruginosa chromosome. Replacement of the wild-type gene with the inactivated gene was verified by Southern analysis and confirmed by lack of elastase antigen on Western blots and lack of activity in liquid assays. The mutant did, however, retain elastolytic activity on elastin plates. This residual activity was abolished by inactivation of lasB in PAO-E64, a lasA-deficient mutant, demonstrating that it was due to the lasA gene product. Northern analysis demonstrated that, like lasB, lasA is transcriptionally controlled by the lasR gene product.