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This report summarizes the application of imaging spectroscopy to the study of biotic components of Yellowstone National Park ecosystems. Maps of vegetation cover and hot-spring microorganisms were generated using spectral-feature analysis of data from the airborne visible and infrared imaging spectrometer (AVIRIS). AVIRIS data were calibrated to surface reflectance using a radiative-transfer model and a ground-calibration target. A spectral library of canopy-reflectance signatures was created by averaging pixels of reflectance data over known occurrences of 27 vegetation cover types in Yellowstone. Distributions of these vegetation types were determined by comparing absorption features of the vegetation in the spectral library with every pixel of the AVIRIS data using continuum removal and spectral analysis in the U.S. Geological Survey’s Tetracorder expert system. Analysis of the chlorophyll- and leaf-water-absorption features (centered near 0.68, 0.98, and 1.20 μm, respectively) allowed accurate identification of vegetation cover types. Conifer cover types of lodgepole pine, whitebark pine, Douglas fir, and a mixed Engelmann spruce/subalpine fir class were spectrally identified and their distributions were mapped in AVIRIS images. Field-reflectance measurements revealed a distinct spectral signature of hot-spring microorganisms. These field measurements were added to the vegetation spectral library, and maps showing the distributions of microbial mats in the geyser basins of Yellowstone were produced.