The aim of this study was to explore biodegradation potential of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in a deep contaminated unsaturated zone over Israel's coastal aquifer. While anaerobic biodegradation potential was observed throughout the profile down to the water table at a depth of 45 m, aerobic biodegradation was limited to the surface of the unsaturated zone. Traces of nitroso-RDX intermediates were detected in the soil samples, indicating possible in situ activity. Polymerase chain reaction and denaturing gradient gel electrophoresis analysis revealed that the microbial population in the soil consisted of protobacteria, but no known RDX degraders were detected. However, a 16S rRNA gene sequence most similar to Sphingomonas sp. was detected at all depths. Biodegradation rates were faster in the surface (0 and 1m) versus deeper soil samples (22 and 45 m) and were not affected under anaerobic conditions by the presence of nitrate, indicating a concurrent reduction of both compounds. RDX half-life in the surface soil was mostly dependent on carbon content and to lesser extent on soil moisture. Biomineralization of RDX to CO(2) was confirmed by incubating surface soil with (14)C-labeled RDX. An aerobic RDX-degrading bacterium, identified as Gordonia sp., was isolated from the soil it degraded RDX aerobically and produced 4-nitro-2,4-diazabutanal. This study, the first to explore RDX biodegradation in the deep vadoze zone, indicates biodegradation potential throughout the profile, which is likely to support natural attenuation.