The 2,4-dichlorophenoxyacetic acid (2,4-D) degrading potential of an agricultural gleyic luvisol from the Dijon area (France) was assessed. 8% of the soil aggregates tested were shown to degrade chain labelled [14C]2,4-D. Mineralization was observed either immediately or after a 4 week lag period. The diversity of the 2,4-D degrading microflora of three aggregates was further investigated. 16S rRNA genes from 68 isolates were therefore amplified, which led to the discrimination of 17 distinguishable RFLP patterns. Instability of the side chain 2,4-D degrading capabilities was observed amongst 25% of the isolates. None of the unstable 2,4-D degraders amplified when the tfdA and tfdB primer sets were used. These strains presented different 16S patterns by the RsaI restriction enzyme and therefore belonged to various microbial species. Amongst the stable 2,4-D degrading isolates, two strains which were isolated from different aggregates did amplify using both tfdA and tfdB primer sets. These strains presented identical tfdA and tfdB PCR-RFLP patterns which were of the A. eutrophus JMP134 (pJP4) and Burkholderia sp RASC type, respectively, but showed different 16S rDNA PCR-RFLP patterns demonstrating that the degrading genes were distributed amongst various bacterial groups prior to any enrichment. Six strains amplified using the tfdB primer set but not using the tfdA primer set. These strains presented either a Burkholderia sp. RASC tfdB pattern or an undescribed new tfdB PCR-RFLP pattern but all shared a common 16S PCR-RFLP pattern. A third group contained stable degraders in which the presence of tfdA and tfdB-like genes could not be detected. These strains presented 12 distinct 16S PCR-RFLP patterns which differed from those observed in the first two groups of stable degraders. Therefore our results demonstrate the involvement of diverse bacterial species and genes in 2,4-D degradation within a soil.