Hydrocarbon spills on Antarctic soils occur mainly near settlements where fuel is stored and aircraft and vehicles are refuelled. To investigate those factors that may preclude hexadecane mineralization activity in long-term hydrocarbon-contaminated soils from the Ross Sea Region, samples were collected from Scott Base, the site of former bases (Cape Evans, Marble Point, Vanda Station), and two oil spill sites in the Wright Valley (Bull Pass and Loop Moraine). The soils had low levels of nitrogen (<0.1% total N) and a high C/N ratio (>24) reflecting hydrocarbon contamination. Following soil water adjustment to 10% (v/w), the influence of nutrient addition (250 mg/kg N added as monoammonium phosphate) and inoculation (spiking with Antarctic soil containing high numbers of hydrocarbon degraders) as required on hexadecane mineralization activity was determined. Hexadecane mineralization activity occurred in contaminated soils from Marble Point, Cape Evans and one sample from Vanda Station without nutrient addition. In contrast soils from Scott Base, Cape Evans, another sample from Vanda Station and Loop Moraine required nutrients, whereas Bull Pass soil required inoculation and nutrients before hexadecane mineralization proceeded. Hydrocarbon degrader numbers were highest in coastal soils from Scott Base and Marble Point (10⁷ per gram) and less prevalent in inland soils from Wright Valley (<10⁵ per gram). The bacterial community structure of the soils differed between sites, but soils from the same sites tended to cluster together more closely, except for those from Vanda Station. Addition of nutrients did not cause large shifts in the soil bacterial communities. Results from this study indicate that hydrocarbon degradation may occur at some sites in summer when water is available. Long-term hydrocarbon-contaminated Antarctic soils may provide a valuable resource of hydrocarbon-degrading bacteria that can serve as inocula for more recent oil spills on land.