Motility is one of the most important traits for rhizosphere colonization by pseudomonads. Despite this importance, motility is severely repressed in the rhizosphere-colonizing strain Pseudomonas fluorescens F113. This bacterium is unable to swarm under laboratory conditions and produce relatively small swimming haloes. However, phenotypic variants with the ability to swarm and producing swimming haloes up to 300% larger than the wild-type strain, arise during rhizosphere colonization. These variants harbour mutations in the genes encoding the GacA/GacS two-component system and in other genes. In order to identify genes and pathways implicated in motility repression, we have used generalized mutagenesis with transposons. Analysis of the mutants has shown that besides the Gac system, the Wsp system and the sadB gene, which have been previously implicated in cyclic di-GMP turnover, are implicated in motility repression: mutants in the gacS, sadB or wspR genes can swarm and produce swimming haloes larger than the wild-type strain. Epistasis analysis has shown that the pathways defined by each of these genes are independent, because double and triple mutants show an additive phenotype. Furthermore, GacS, SadB and WspR act at different levels. Expression of the fleQ gene, encoding the master regulator of flagella synthesis is higher in the gacS- and sadB- backgrounds than in the wild-type strain and this differential expression is reflected by a higher secretion of the flagellin protein FliC. Conversely, no differences in fleQ expression or FliC secretion were observed between the wild-type strain and the wspR- mutant.