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This paper synthesizes results of a biological inventory of a 219-km2 nature preserve in the central Niobrara Valley of Nebraska. The two-year inventory documented the presence of 80 species of mosses and liverworts, 581 of vascular plants, 130 of aquatic Invertebrates and 278 of vertebrate animals. Comparison with distribution records from four other counties in Nebraska shows that the Niobrara study site has higher vertebrate species diversity despite its smaller area. Although the study area is in the central grasslands of North America, many of the species here are more typical of forest biomes far to the east, north and west. Local geohydrological features and historical events contribute to this unique mix of species and communities.