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To date, all studies of aflatoxin B-1 (AFB(1)) transformation in soil or in purified mineral systems have identified aflatoxins B-2 (AFB(2)) and G(2) (AFG(2)) as the primary transformation products. However, identification in these studies was made using thin layer chromatography which has relatively low resolution, and these studies did not identify a viable mechanism by which such transformations would occur. Further, the use of methanol as the solvent delivery vehicle in these studies may have contributed to formation of artifactual transformation products. In this study, we investigated the role of the solvent vehicle in the transformation of AFB(1) in soil. To do this, we spiked soils with AFB(1) dissolved in water (93:7, water/methanol) or methanol and used HPLC-UV and HPLC-MS to identify the transformation products. Contrasting previous published reports, we did not detect AFB(2) or AFG(2). In an aqueous-soil environment, we identified aflatoxin B-2a (AFB(2a)) as the single major transformation product. We propose that AFB(2a) is formed from hydrolysis of AFB(1) with the soil acting as an acid catalyst. Alternatively, when methanol was used, we identified methoxy aflatoxin species likely formed via acid-catalyzed addition of methanol to AFB(1). These results suggest that where soil moisture is adequate, AFB(1) is hydrolyzed to AFB(2a) and that reactive organic solvents should be avoided when replicating natural conditions to study the fate of AFB(1) in soil.