Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common contaminant at explosives production sites. Here, we report on the use of compound-specific isotope analysis of RDX to obtain delta(15)N and delta(18)O enrichment factors during biodegradation in batch cultures. A new preparation method has been developed based on RDX purification using thin-layer chromatography. RDX is then subjected to an elemental analyzer coupled with an isotope-ratio mass spectrometer (EA-IRMS). The precision of the method shows standard deviations of 0.13% per hundred and 1.18% per hundred for delta(15)N and delta(18)O, respectively, whereas the accuracy of the method has been checked routinely, adhering to external standards. The method was applied to RDX samples subjected to biodegradation under aerobic or anaerobic conditions. Enrichment factors under aerobic conditions were -2.1% per hundred and -1.7% per hundred for delta(15)N and delta(18)O, respectively, and under anaerobic conditions, -5.0% per hundred and -5.3% per hundred for delta(15)N and delta(18)O, respectively. The results of this study provide a tool for monitoring natural attenuation of RDX in a contaminated environment.