Rapid physiological profiling of heterotrophic microbial communities enables intensive analysis of the factors affecting activity in aerobic habitats, such as soil. Previous methods for performing such profiling were severely limited due to enrichment bias and inflexibility in incubation conditions. We tested a new physiological profiling approach based on a microtiter plate oxygen sensor system (Becton Dickinson Oxygen Biosensor System (BDOBS)), which allows for testing of lower substrate addition (i.e., lower enrichment potential) and manipulation of physiochemical assay conditions, such as pH and nutrients. Soil microbial communities associated with a scrub-oak forest ecosystem on Merritt Island Wildlife Refuge in central Florida, USA, were studied in order to evaluate microbial activity in a nutrient poor soil and to provide baseline data on the site for subsequent evaluation of the effects of elevated CO(2) on ecosystem function. The spatial variation in physiological activity amongst different habitats (litter, bulk soil, and rhizosphere) was examined as a function of adaptation to local resources (i.e., water soluble extracts of roots and leaf litter) and the degree of N and P limitation. All the communities were primarily N-limited, with a secondary P limitation, which was greater in the rhizosphere and bulk soil. The litter community showed greater overall oxygen consumption when exposed to litter extracts relative to the rhizosphere or soil, suggesting acclimation toward greater use of the mixed substrates in the extract. Root extracts were readily used by communities from all the habitats with no habitat specific acclimation observed. A priming effect was detected in all habitats; addition of glucose caused a significant increase in the use of soil organic carbon. Response to added glucose was only observed with N and P addition, suggesting that C may be lost to the groundwater from these porous soils because nutrient limitation prevents C immobilization.