U(VI) accumulation by the acidothermophilic archaeon Sulfolobus acidocaldarius at a moderate acidic pH of 4.5 was investigated. This pH value is relevant for some heavy metal and uranium polluted environments where populations of S. acidocaldarius were found to persist. We demonstrate that U(VI) is rapidly complexed by the archaeal cells. A combination of X-ray absorption spectroscopy and time-resolved laser-induced fluorescence spectroscopy revealed that at pH 4.5 organic phosphate and carboxylic groups are involved in the U(VI) complexation. These results are in contrast to those published for most bacteria which at this pH precipitate U(VI) mainly in inorganic uranyl phosphate phases. As demonstrated by TEM only a limited part of the added U(VI) was biomineralized extracellularly in the case of the studied archaeon. Most of the U(VI) accumulates were localized in a form of intracellular deposits which were associated with the inner side of the cytoplasma membrane. Observed differences in U(VI) bioaccumulation between the studied archaeon and bacteria can be explained by the significant differences in their cell wall structures as well as by their different physiological characteristics.