The bacterium Herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. However, the mechanisms involved in the interaction between H. seropedicae and these plants are not completely characterized. We investigated the attachment of Herbaspirillum to maize roots and the invasion of the roots by this bacterium using H. seropedicae strain SMR1 transformed with the suicide plasmid pUTKandsRed, which carries a mini-Tn5 transposon containing the gene for the Discosoma red fluorescent protein (Dsred) constitutively expressed together with the kanamycin resistance gene. Integration of the mini-Tn5 into the bacterial chromosome yielded the mutant H. seropedicae strain RAM4 which was capable of expressing Dsred and could be observed on and inside fresh maize root samples. Confocal microscopy of maize roots inoculated with H. seropedicae three days after germination showed that H. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days.