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Cross-calibration of satellite sensors permits the quantitative comparison of measurements obtained from different Earth Observing (EO) systems. Cross-calibration studies usually use simultaneous or near-simultaneous observations from several spaceborne sensors to develop band-by-band relationships through regression analysis. The investigation described in this paper focuses on evaluation of the uncertainties inherent in the cross-calibration process, including contributions due to different spectral responses, spectral resolution, spectral filter shift, geometric misregistrations, and spatial resolutions. The hyperspectral data from the Environmental Satellite SCanning Imaging Absorption SpectroMeter for Atmospheric CartograpHY and the EO-1 Hyperion, along with the relative spectral responses (RSRs) from the Landsat 7 Enhanced Thematic Mapper (TM) Plus and the Terra Moderate Resolution Imaging Spectroradiometer sensors, were used for the spectral uncertainty study. The data from Landsat 5 TM over five representative land cover types (desert, rangeland, grassland, deciduous forest, and coniferous forest) were used for the geometric misregistrations and spatial-resolution study. The spectral resolution uncertainty was found to be within 0.25%, spectral filter shift within 2.5%, geometric misregistrations within 0.35%, and spatial-resolution effects within 0.1% for the Libya 4 site. The one-sigma uncertainties presented in this paper are uncorrelated, and therefore, the uncertainties can be summed orthogonally. Furthermore, an overall total uncertainty was developed. In general, the results suggested that the spectral uncertainty is more dominant compared to other uncertainties presented in this paper. Therefore, the effect of the sensor RSR differences needs to be quantified and compensated to avoid large uncertainties in cross-calibration results.