ABSTRACT Cyclic lipopeptides (CLPs) are produced by many Pseudomonas species and have several biological functions, including a role in surface motility, biofilm formation, virulence, and antimicrobial activity. This study focused on the diversity and role of LuxR-type transcriptional regulators in CLP biosynthesis in Pseudomonas species and, specifically, viscosin production by Pseudomonas fluorescens strain SBW25. Phylogenetic analyses showed that CLP biosynthesis genes in Pseudomonas strains are flanked by LuxR-type regulators that contain a DNA-binding helix-turn-helix domain but lack N -acylhomoserine lactone-binding or response regulator domains. For SBW25, site-directed mutagenesis of the genes coding for either of the two identified LuxR-type regulators, designated ViscAR and ViscBCR, strongly reduced transcript levels of the viscABC biosynthesis genes and resulted in a loss of viscosin production. Expression analyses further showed that a mutation in either viscAR or viscBCR did not substantially (change of <2.5-fold) affect transcription of the other regulator. Transformation of the Δ viscAR mutant of SBW25 with a LuxR-type regulatory gene from P. fluorescens strain SS101 that produces massetolide, a CLP structurally related to viscosin, restored transcription of the viscABC genes and viscosin production. The results further showed that a functional viscAR gene was required for heterologous expression of the massetolide biosynthesis genes of strain SS101 in strain SBW25, leading to the production of both viscosin and massetolide. Collectively, these results indicate that the regulators flanking the CLP biosynthesis genes in Pseudomonas species represent a unique LuxR subfamily of proteins and that viscosin biosynthesis in P. fluorescens SBW25 is controlled by two LuxR-type transcriptional regulators.