Aquifer microbes in the 300 Area of the Hanford Site in southeastern Washington State, USA, are located in an oligotrophic environment and are periodically exposed to U(VI) concentrations that can range up to 10 μM in small sediment fractures. Assays of (3)H-leucine incorporation indicated that both sediment-associated and planktonic microbes were metabolically active, and that organic C was growth-limiting in the sediments. Although bacteria suspended in native groundwater retained high activity when exposed to 100 μM U(VI), they were inhibited by U(VI) <1 μM in synthetic groundwater that lacked added bicarbonate. Chemical speciation modeling suggested that positively charged species and particularly (UO2)3(OH)5 (+) rose in concentration as more U(VI) was added to synthetic groundwater, but that carbonate complexes dominated U(VI) speciation in natural groundwater. U toxicity was relieved when increasing amounts of bicarbonate were added to synthetic groundwater containing 4.5 μM U(VI). Pertechnetate, an oxyanion that is another contaminant of concern at the Hanford Site, was not toxic to groundwater microbes at concentrations up to 125 μM.