Previous studies demonstrated the role of the O-antigenic side chain of the outer membrane lipopolysaccharides of Pseudomonas fluorescens strain WCS417r in its ability to induce resistance against fusarium wilt disease. The present study compared the colonization of tomato roots (Lycopersicon esculentum Mill.) by P. fluorescens strain WCS417r with that of a mutant of this strain, lacking the O-antigenic side chain of its outer membrane lipopolysaccharides (OA−). Special attention was paid to colonization within root tissues and changes of the root ultrastructure induced by the bacterial strains. Experiments were performed with tomato plants rooted in rock wool and tomato seedlings grown on agar. Numeration of colony-forming units showed that wild-type strain WCS417r colonized the root interior of tomato grown in rock wool to a higher extent than its OA− mutant B4, whereas colonization of the rhizoplane did not differ between the two. Light microscopy observations of cross sections of roots grown on agar showed that (i) colonization of the root interior by both wild-type and OA− mutant was inter- and intracellular, (ii) colonization of the internal root tissues mainly occurred if the Pseudomonas strains were inoculated at sites of lateral root emergence, (iii) the wild type colonized the root interior more frequently than did its OA− mutant. Thickening of the cortical cell walls, in reaction to the bacterial colonization of epidermal or hypodermal cells, or cortical intercellular spaces, suggested that local defence reactions of the plant occurred. Altogether, these results indicate the involvement of the O-antigenic side chain of the outer membrane lipopolysaccharides in the endophytic colonization of the tomato root by P. fluorescens WCS417r, and lead us to hypothesize that induction of disease resistance by strain WCS417r might be related to the extent of colonization of the internal root tissues.