Pseudomonas fluorescens CHA0 protects plants from damage caused by several soilborne fungi. In this work, immunofluorescence microscopy was used to investigate the colonization of tobacco roots by CHA0 and its physical relationship with the black root rot fungus Thielaviopsis basicola. The pseudomonad colonized the rhizoplane shortly after planting of tobacco seedlings in sterile soil microcosms, in which it had been introduced as soil inoculant. CHA0 was found between and inside cells in the epidermis and the cortex, as well as in the xylem vessels, within 4-7 days after planting of seedlings. The presence of CHA0 delayed the colonization of the interior of tobacco roots by T. basicola compared with the treatment in which only the fungus had been inoculated. Likewise, the pseudomonad reduced the extent of black root rot from 82% to 28%. However, CHA0 was seldom found in contact with the mycelium of T. basicola or in its vicinity, indicating that direct colonization of the mycelium of T. basicola by CHA0 was not required for protection of tobacco against black root rot. Overall, the results suggest that the interior of the root is a key site for implementation of the strain's biocontrol activity against soilborne plant-pathogenic fungi.