To determine whether the diversity of phenanthrene-degrading bacteria in an aged polycyclic aromatic hydrocarbon (PAH) contaminated soil is affected by the addition of plant root exudates, DNA stable isotope probing (SIP) was used. Microcosms of soil with and without addition of ryegrass exudates and with 13C-labelled phenanthrene (PHE) were monitored over 12 days. PHE degradation was slightly delayed in the presence of added exudate after 4 days of incubation. After 12 days, 68% of added PHE disappeared both with and without exudate. Carbon balance using isotopic analyses indicated that a part of the 13C-PHE was not totally mineralized as 13CO2 but unidentified 13C-compounds (i.e. 13C-PHE or 13C-labelled metabolites) were trapped into the soil matrix. Temporal thermal gradient gel electrophoresis (TTGE) analyses of 16S rRNA genes were performed on recovered 13C-enriched DNA fractions. 16S rRNA gene banding showed the impact of root exudates on diversity of PHE-degrading bacteria. With PHE as a fresh sole carbon source, Pseudoxanthomonas sp. and Microbacterium sp. were the major PHE degraders, while in the presence of exudates, Pseudomonas sp. and Arthrobacter sp. were favoured. These two different PHE-degrading bacterial populations were also distinguished through detection of PAH-ring hydroxylating dioxygenase (PAH-RHDa) genes by real-time PCR. Root exudates favoured the development of a higher diversity of bacteria and increased the abundance of bacteria containing known PAH-RHDa genes.