Nutrient enrichment of wetland soils may alter patterns of heterotrophic microbial activity (HMA), which influences organic matter accumulation and decomposition. Patterns in the utilization of C-substrates by heterotrophic microbial communities in response to changes in environmental factors may serve as indicators of changes in the trophic status of wetlands. The response of the heterotrophic microbial community to C-substrates was measured in detritus and soil (0-10 cm) for eutrophic, transitional, and oligotrophic sites along a nutrient enrichment gradient in Water Conservation Area 2a (WCA-2a) of the Florida Everglades. The short-term response (2-d incubation) to C-substrates (alcohols, amino acids, carboxylic acids, and polysaccharides) was measured as CO2 production. The nutrient gradient was characterized by decreasing P concentrations and microbial biomass from eutrophic to oligotrophic sites. Basal respiration was 73% higher at eutrophic than oligotrophic sites, and 41% higher in detritus than underlying soil. Patterns of C-substrate utilization varied with greater utilization of substrates at the eutrophic site, presumably a result of higher levels of microbial biomass and inorganic nutrients compared to the oligotrophic site. Substrate-induced respiration (SIR) of detritus was 25, 45, and 42% greater for polysaccharides than other C-substrates at the eutrophic, transitional, and oligotrophic sites, respectively. Likewise, SIR at the eutrophic, transitional, and oligotrophic sites was 0, 40, and 39%, respectively, greater for detritus amended with carboxylic acids than with amino acids and alcohols. Polysaccharides dominated HMA profiles at all sites along the nutrient gradient in detritus. The transitional site was characterized by carboxylic acids and alcohols, while the HMA profile at the oligotrophic site was dominated by carboxylic acids. The C-substrates enhanced CO2 production at all sites suggesting that labile organic C was a limiting factor to HMA. Patterns of HMA along the nutrient enrichment gradient provided insight into the microbial response to changes in environmental conditions.