Aim: Characterization of an anaerobic thermophilic bacterium and subcellular localization of its Cr(VI)-reducing activity for potential bioremediation applications. Methods and Results: 16S rRNA gene sequence-based analyses of bacterial strains isolated from sediment samples of a Bakreshwar (India) hot spring, enriched anaerobically in iron-reducing medium, found them to be 86-96% similar to reported Thermoanaerobacter strains. The most efficient iron reducer among these, BSB-33, could also reduce Cr(VI) at an optimum temperature of 60 degrees C and pH 6.5. Filtered culture medium could reduce Cr(VI) but not Fe(III). Cell-free extracts reduced Cr(VI) inefficiently under aerobic conditions but efficiently anaerobically. Fractionation of the cell-free extracts showed that chromium reduction activity was present in both the cytoplasm and membrane. Conclusions: BSB-33 reduced Fe(III) and Cr(VI) anaerobically at 60 degrees C optimally. After fractionation, the reducing activity of Cr(VI) was found in both cytoplasmic and membrane fractions. Significance and Impact of the Study: To the best of our knowledge, this is the first systematic study of anaerobic Cr(VI) reduction by a gram-positive thermophilic micro-organism and, in contrast to our results, none of the earlier reports has mentioned Cr(VI)-reducing activity to be present both in the cytoplasm and membrane of an organism. The strain may offer itself as a potential candidate for bioremediation.