- The explosive Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is known to be degraded aerobically by various isolates of the Rhodococcus species, with denitration being the key step, mediated by Cytochrome P450. Our study aimed at gaining insight into the RDX degradation mechanism by Rhodococcus species and comparing isotope effects associated with RDX degradation by distinct Rhodococcus strains. For these purposes, enrichment in 13C and 15N isotopes throughout RDX denitration was studied for three distinct Rhodococcus strains, isolated from soil and groundwater in an RDX-contaminated site. The observable 15N enrichment throughout the reaction, together with minor 13C enrichment, suggests that N–N bond cleavage is likely to be the key rate-limiting step in the reaction. The similarity in the kinetic 15N isotope effect between the three tested strains suggests that either isotope-masking effects are negligible, or are of a similar extent for all tested strains. The lack of variability in the kinetic 15N isotope effect allows the interpretation of environmental studies with greater confidence.