In the Paso del Norte region, both rural and urban border communities are exposed to trace metals from industry, atmospheric fallout from smelting, combustion of petroleum products, and the reclamation of wastewaters for agricultural use. Chemical fractionation methods to determine the bioavailability/bio-accessibility of metals in soils, particularly under alkaline conditions have been inconsistent to poor indicators of predicting trace metal accumulation in plants. Consequently, risk assessment of contaminated soil to animals and humans has been limited. In vitro gastrointestinal (IVG) and physiologically based extraction test (PBET) methods may provide better estimates of trace metal bioavailability and human risk. The objective of this study was to compare chemical fractionation methods with modified IVG and PBET methods to predict the bio-accessibility of cadmium (Cd), chromium (Cr), nickel (Ni) and lead (Pb) in moderately alkaline soils with and without biosolids amendments in the Juarez Valley of Mexico. The exposure pathway for this study was; untreated effluents for irrigation – soil incorporated biosolids - metal contaminated soil – oat forage – sheep. Selected trace metals in soil and biosolids were chemically extracted using total and sequential metal extraction protocols, total acid extraction in plant tissue and in sheep tissues (muscle, liver, and kidneys). Soil samples were re-extracted using modified IVG and PBET methods. Sequential fractionation of selected trace elements in soil did not reflect metal accumulation in oat forage or in sheep kidney, liver, or muscle tissues. The validity of metal bio-accessibility using in vitro methods was dependent on the metal species. In comparison to actual metal concentrations found in sheep tissues, Pb bio-accessibility was consistently overestimated, Ni concentrations were closely predicted, and Cd and Cr bio-accessibility was dependent on the extraction method. Ruminate (sheep) digestion and absorption of metals may account for discrepancies between actual metal accumulation in sheep tissue and bio-asscessibility determinations developed for monogastrics.