The aim of this study was to investigate the supposed vertical diel migration and the accompanying physiology of Beggiatoa bacteria from hypersaline microbial mats. We combined microsensor, stable-isotope, and molecular techniques to clarify the phylogeny and physiology of the most dominant species inhabiting mats of the natural hypersaline Lake Chiprana, Spain. The most dominant morphotype had a filament diameter of 6 to 8 microm and a length varying from 1 to >10 mm. Phylogenetic analysis by 16S rRNA gene comparison revealed that this type appeared to be most closely related (91% sequence identity) to the narrow (4-microm diameter) nonvacuolated marine strain MS-81-6. Stable-isotope analysis showed that the Lake Chiprana species could store nitrate intracellularly to 40 mM. The presence of large intracellular vacuoles was confirmed by fluorescein isothiocyanate staining and subsequent confocal microscopy. In illuminated mats, their highest abundance was found at a depth of 8 mm, where oxygen and sulfide co-occurred. However, in the dark, the highest Beggiatoa densities occurred at 7 mm, and the whole population was present in the anoxic zone of the mat. Our findings suggest that hypersaline Beggiatoa bacteria oxidize sulfide with oxygen under light conditions and with internally stored nitrate under dark conditions. It was concluded that nitrate storage by Beggiatoa is an optimal strategy to both occupy the suboxic zones in sulfidic sediments and survive the dark periods in phototrophic mats.