Herbaceous communities located within forest openings increase plant species diversity of forests in the Knobs Region of Kentucky. Although these grass-dominated communities are protected and managed for rare plant species conservation, it is unclear how soil conditions may delineate the grassland-forest boundary. We compared soil chemical and physical properties and soil nitrogen pools and transformations of three forest openings with those in the surrounding forest and the wooded edge separating the grassland and forest communities. Soil pH, extractable cations, bulk density and silt content were higher in openings compared to forest soils and extractable phosphorus was lower. In forest-opening grasslands, total soil nitrogen and the availability and production of plant-available N were 25 to 50% of levels in adjacent forest. Soils of the woody edge were similar to the surrounding forest. Our results suggest that the presence of these calcareous glade openings is highly correlated with edaphic conditions, so that efforts to expand rare plant habitat may be constrained by the distinct soils of these forest openings.