Complete removal of plants and soil to exposed bedrock, in order to eradicate the Hole-in-the-Donut (HID) region of the Everglades National Park, FL, of exotic invasive plants, presented the opportunity to monitor the redevelopment of soil and the associated microbial communities along a short-term restoration chronosequence. Sampling plots were established for sites restored in 1989, 1997, 2000, 2001, and 2003. The goal of this study was to characterize the activity and diversity of denitrifying bacterial populations in developing HID soils in an effort to understand changes in nitrogen (N) cycling during short-term primary succession. Denitrifying enzyme activity (DEA) was detected in soils from all sites, indicating a potential for N loss via denitrification. However, no correlation between DEA and time since disturbance was observed. Diversity of bacterial denitrifiers in soils was characterized by sequence analysis of nitrite reductase genes (nirK and nirS) in DNA extracts from soils ranging in nitrate concentrations from 1.8 to 7.8 mg kg ؊1 . High levels of diversity were observed in both nirK and nirS clone libraries. Statistical analyses of clone libraries suggest a different response of nirS-and nirK-type denitrifiers to factors associated with soil redevelopment. nirS populations demonstrated a linear pattern of succession, with individual lineages represented at each site, while multiple levels of analysis suggest nirK populations respond in a grouped pattern. These findings suggest that nirK communities are more sensitive than nirS communities to environmental gradients in these soils.The Hole-in-the-Donut (HID) is a 4,000-ha region within Everglades National Park, FL. The area originally consisted of oligotrophic sawgrass (Cladium jamaicense Crantz) prairies and short-hydroperiod pinelands but was converted to farmland during the early part of the twentieth century (4, 14). As preparation for the conversion to agriculture, intensive rock plowing pulverized underlying limestone bedrock and created coarse-textured, well-drained soil suitable for vegetable production (12). Farming in the HID ceased in 1976, and the abandoned farmland in this area was soon invaded by Schinus terebinthifolius Raddi (Brazilian pepper), an exotic shrub native to South America.Everglades National Park initiated efforts to restore the HID in 1996 by complete removal of all plants and much of the soil down to bedrock. An individual plot within the HID is cleared at one time, such that plots representing a chronosequence after clearing are present at one time (4). Following clearing, individual plots are left undisturbed to allow natural establishment of microbial communities and colonization by native wetland plants. This staggered approach to clearing provides an excellent opportunity to study the redevelopment of soil, microbial communities, and ecosystem processes over a short-term chronosequence in this wetland.A previous report on succession of methanogenic communities at this site indicated shifts within species and act...