The potential for removing uranium from contaminated groundwater by stimulating the activity of U(VI)-reducing microorganisms was evaluated in aquifer sediments collected near a uranium mill tailings pile. Added acetate or glucose stimulated U(VI) removal more effectively than other added electron donors such as lactate, benzoate, or formate. U(VI) was removed concurrently with the reduction of Fe(III) and prior to sulfate reduction. With the addition of acetate, initial concentrations of approximately 10 muM U(VI) were reduced to less than 1 muM within 15 days. The addition of humic acids or the humics analog, anthraquinone-2,6-disulfonate (AQDS), as electron shuttles, stimulated the rate of Fe(III) reduction, but not U(VI) reduction. This was because reduced humics and AQDS reduced U(VI) much more slowly than Fe(III). Neither sulfide, reduced AQDS, nor Fe(II) abiotically reduced U(VI) in the sediments. These results suggest that controlled addition of low concentrations of electron donor, particularly acetate, to uranium-contaminated aquifers can stimulate microbial U(VI) reduction and promote the removal of soluble U(VI) from contaminated groundwater without the production of toxic sulfides. The finding that microbial U(VI) reduction was the primary mechanism for U(VI) removal emphasizes the importance of understanding the ecophysiology of U(VI)-reducing microorganisms in the subsurface, both in designing remediation strategies for uranium contamination and in modeling the uranium geochemistry of ore body formation.