The bioreduction of soluble hexavalent uranium (U-VI) to insoluble tetravalent uranium (U-IV) is an attractive bioremediation strategy for the clean-up of contaminated groundwater. High levels of the common occurring co-contaminant, nitrate (NO3-), can potentially interfere with uranium bioremediation. In this study, treatment of a synthetic groundwater containing a mixture of NO3- and U-VI was investigated in a sulfur-limestone autotrophic denitrifying (SLAD) bioreactor that was coupled in series with a bioreactor packed with zero-valent iron (Fe-0, ZVI) and sand. An additional aim of the study was to explore the possible role of biological activity in enhancing the reduction of U-VI by Fe-0. The SLAD reactor removed NO3- efficiently (99.8%) at loadings of up to 20 mmol NO3- L-r(-1) d(-1), with near stoichiometric conversion to benign dinitrogen gas (N-2). The ZVI bioreactor subsequently removed uranium (99.8%) at high (0.22mM) and low (0.02mM) influent concentrations of the radio-nuclide. Aqueous uranium was reliably eliminated to below the maximum contaminant level of 30 mu g L-1 (0.13 mu M) when the ZVI reactor was operated at average empty bed hydraulic retention times as low as 2.3 h, demonstrating the feasibility of the sequential treatment strategy in packed bed bioreactors. Sequential extraction of the ZVI reactor packing confirmed that uranium was immobilized as U-IV. Uranium removal was enhanced by microbial activity as confirmed by the increased rate of uranium removal in batch assays inoculated with effluent from the ZVI bioreactor and spiked with Fe-0 compared to abiotic controls. Biotechnol. Bioeng. 2010;107: 933-942. (C) 2010 Wiley Periodicals, Inc.