N-Octanoyl-L-homoserine lactone (C8-HSL) is an acyl-homoserine-lactone signal utilized in the quorum-sensing (QS) systems of Burkholderia cenocepacia and other bacterial species. Although also produced by Pseudomonas aeruginosa, its role in this species has not been elucidated. Here, we report that C8-HSL modulated antibiotic resistance and pyocyanin production in a P. aeruginosa efflux pump-deficient mutant. The rhl/las quorum-sensing system and qscR gene were both shown to be nonessential in the C8-HSL-induced changes in ofloxacin resistance, suggesting that P. aeruginosa possesses a distinct pathway to respond to C8-HSL.