Enhanced mobilization of potentially toxic trace elements (PTTE) is well documented for contaminated floodplains, wetlands, and rice paddies. Limited information is available on flooding-induced PTTE release from uncontaminated agricultural soils from temperate regions. We conducted an incubation study with simulated flooding using calcareous, uncontaminated agricultural soils to assess the release dynamics of a few PTTE and to identify the controlling factors. Packed soils were flooded, and soil redox potential (Eh), pH, total dissolved metals, and sulfur (S) concentrations in pore water and floodwater were measured for 8 wk. Pore water arsenic (As) and nickel (Ni) concentrations were initially low, but significantly increased with flooding by 12- and 6-fold, respectively. Copper (Cu) concentration declined with flooding, whereas zinc (Zn) showed no consistent trend. Arsenic and Ni concentrations correlated negatively with Eh, and positively with other redox-sensitive elements, whereas Cu behaved in a manner opposite to that of As and Ni, however, correlating positively with S. Pore water Zn correlated negatively with pH and Ca, and positively with Al. Principal component analysis confirmed the role of Eh on As, Ni, and Cu release from flooded soils and the pH dependency on Zn release. Prolonged flooding released environmentally significant quantities of some PTTE from uncontaminated agricultural soils.