As components of a Pseudomonas aeruginosa quorum-sensing system, LasR and PAI-1 globally regulate expression of multiple virulence determinants, as well as the second P. aeruginosa quorum-sensing system. To date, no information exists on negative regulation of the quorum-sensing cascade in P. aeruginosa. Here we describe a novel gene, rsaL, which is located downstream from lasR and transcribed antisense relative to lasR. In P. aeruginosa, overexpression of rsaL results in reduced lasB expression and decreased elastase activity. With the use of a six-His protein fusion system, we demonstrate that rsaL encodes an 11-kDa protein. Direct quantitation of PAI-1 levels in cultures and studies utilizing Escherichia coli lambda lysogens carrying lacZ transcriptional fusions reveal that RsaL specifically represses transcription of the PAI-1 autoinducer synthase gene, lasI. RsaL's repressive effect on lasI and the associated decrease in elastase activity have important implications for the expression of all LasR-PAI-1-dependent virulence genes and the overall pathogenicity of P. aeruginosa.