Uranium partitioning in soils collected from an acid and U impacted sandy Coastal Plain aquifer at the Savannah River Site (SRS) was investigated. The influences of hydrologic regime (vadose zone or saturated zone), proximity to the source input (impacted or unimpacted soils), and soil weathering (field or laboratory-spiked soils) on the environmental availability U were examined. Environmental availability (availability for groundwater transport) was operationally defined using a sequential extraction technique and was applied to vadose zone, saturated zone, and background soils. For saturated zone locations, matched porewater–soil sets of field samples were collected, and data generated from these samples were used to examine U partitioning under field conditions. Laboratory batch sorption studies of uranyl ion to background soils were conducted as a function of pH. Subsequently, the soil used in the sorption study was subjected to sequential extraction to investigate the environmental availability in laboratory spiked samples. Based on sequential extraction behavior of U-impacted soils and background soils and the acidic plume chemistry, U concentrations in the first three sequential extraction steps [deionized water, CaCl2, and acetic acid/Ca(NO3)2] were operationally defined as available, and the final two extraction steps (crystalline iron oxide and residual extraction steps) were operationally defined as unavailable. Based on this operational definition, soils impacted by the acidic U plume exhibited a greater fraction of available and total U. Vadose-zone soils had a smaller fraction of available U than corresponding saturated zone samples. Sequential extractions of U sorbed to background soils in a short-term laboratory experiment showed greater U availability compared with field soils collected within the contaminant plume. Field-derived Kd values ranged from 0.1 to 300 L kg−1 and were highly correlated with porewater pH.