Marsupials have lower resting metabolic rates than placental mammals, but it is not clear whether particular species can extend this energetic advantage to locomotion. Some active marsupials have a low cost of locomotion, but other more sedentary species, such as the Virginia opossum, appear to run very inefficiently. Steady-state rates of O2 consumption (VO2) and CO2 production (VCO2) were measured at rest and during horizontal treadmill exercise in wild-caught, trained opossums. Average daily VO2 in in undisturbed animals was 7.73 +/- 0.40 ml O2 kg-1 min-1 (5.67 +/- 0.20 ml O2 kg-1 min-1 during light and 9.84 +/- 0.81 ml O2 kg-1 min-1 during dark hours, mean +/- S.E.M., N = 6). Net cost of locomotion ranged between 6.16 and 8.99 J kg-1 s-1 as speed increased and was always higher than for an average mammal of equivalent mass. Net cost of transport decreased as speed increased and was 15-80% higher than for an average mammal. During aerobic locomotion, most of the energy was provided by carbohydrate oxidation, which accounted for 60-95% of VO2 as speed increased. Glycogen and triglyceride reserves were quantified in the major storage depots to estimate potential survival time and travelling distance. Enough metabolic fuel was stored to survive for at least 1 week without eating, and 95% of this energy was in adipose tissue triglycerides. However, maximal travelling distance was less than 2 km because opossum locomotion is mainly supported by carbohydrate reserves, which represented only 4% of the available energy. We conclude that aerobic, ground locomotion of Virginia opossums is associated with two major energetic handicaps because their particularly high cost of transport and the nature of the main oxidative fuel they consume are both incompatible with prolonged locomotion.