<jats:title>ABSTRACT</jats:title><jats:p>Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various<jats:italic>Pseudomonas</jats:italic>strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in<jats:italic>P. fluorescens</jats:italic>SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated<jats:italic>massA</jats:italic>,<jats:italic>massB</jats:italic>, and<jats:italic>massC</jats:italic>, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the<jats:italic>mass</jats:italic>genes contained several genes found in other<jats:italic>Pseudomonas</jats:italic>CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most<jats:italic>Pseudomonas</jats:italic>CLP gene clusters known to date, the<jats:italic>mass</jats:italic>genes are not physically linked but are organized in two separate clusters, with<jats:italic>massA</jats:italic>disconnected from<jats:italic>massB</jats:italic>and<jats:italic>massC</jats:italic>. Quantitative real-time PCR analysis indicated that transcription of<jats:italic>massC</jats:italic>is strongly reduced when<jats:italic>massB</jats:italic>is mutated, suggesting that these two genes function in an operon, whereas transcription of<jats:italic>massA</jats:italic>is independent of<jats:italic>massBC</jats:italic>and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by<jats:italic>N</jats:italic>-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of<jats:italic>P. fluorescens</jats:italic>SS101 and plays an important role in biofilm formation.