During colonization of the alfalfa rhizosphere, Pseudomonas fluorescens F113 undergoes phenotypic variation, resulting in the appearance of colonies with different morphology. Among phenotypic variants, three isolates, C, F, and S were selected, with the C variant showing colony morphology identical to that of the inoculated wild-type strain and F and S having a translucent and diffuse morphology. Phenotypic variants F and S were shown to preferentially colonize distal parts of the roots and showed alterations in motility, swimming faster than the C variant and swarming under conditions that did not allow swarming of the C variant. The motility behavior correlated with overproduction of the fliC-encoded protein flagellin but not with hyperflagellation. Flagella of the F and S variants were several times longer than those of the C variant, and overproduction of flagellin was regulated at the transcriptional level. Variant F showed alterations in traits that have been shown to be important for rhizosphere colonization, such as siderophore, cyanide, and exoprotease production, and these phenotypes were complemented by a cloned gacA. Sequence analysis of the gacA alelle in variant F suggested selection of the phenotype in the rhizosphere. Variant F was also affected in other phenotypes, such as lipopolysaccharide structure and flocculation in unshaken liquid medium, which were not complemented by the gacA or gacS gene. Mutation of the F113 sss gene, encoding a site-specific recombinase, showed that most of the phenotypic variation was due to the activity of this recombinase, indicating that phase variation occurs during rhizosphere colonization.