Genetic bioaugmentation, in which bacteria harboring conjugative plasmids provide catabolic functions, is a promising strategy to restore dioxin-contaminated environments. Here we examined the conjugative transfer of the dioxin-catabolic plasmids pDF01 and pDF02 harbored by Rhodococcus sp. strain p52. A mating experiment using strain p52 as a donor showed that pDF01 and pDF02 were concomitantly and conjugatively transferred from strain p52 to a Pseudomonas aeruginosa recipient at a conjugation frequency of 3 × 10-4 colonies per recipient. pDF01 and pDF02 were isolated from the P. aeruginosa transconjugant and identified by Southern hybridization, and they were localized in the transconjugant cells by fluorescence in situ hybridization. Moreover, the catabolic plasmids functioned in the transconjugant, which gained the ability to use dibenzofuran and chlorodibenzofuran for growth, and they were maintained in 50% of the transconjugant cells for 30 generations without selective pressure. Furthermore, conjugative transfer of the catabolic plasmids to activated sludge bacteria was detected. Sequencing of pDF01 and pDF02 revealed the genetic basis for the plasmids' conjugative transfer and stable maintenance, as well as their cooperation during dioxin catabolism. Therefore, strain p52 harboring pDF01 and pDF02 has potential for genetic bioaugmentation in dioxin-contaminated environments.