A borehole draining a water-bearing dyke fracture at3.2-km depth in a South African Au mine was isolatedfrom the open mine environment. Geochemical, stable isotopic, nucleic acid-based, and phospholipid fatty acid (PLFA) analyses were employed as culture- independent means for assessing shifts in the microbial community and habitat as the system equilibrated with the native rock-water environment. Over a two-month period, the pH increased from 5.5 to 7.4, concurrent with a drop in pe from —2 to -3. Whereas rDNAs related to Desulfotomaculum spp. represented the major clone type encountered throughout, lipid biomarker profiling along with 16S rDNA clone library and terminal restriction fragment length polymorphism (T-RFLP) analyses indicated the emergence of other Gram-positive and deeply-branching lineages in samples during the later stages of the equilibration period. A biofilm that formed on the mine wall below the borehole produced abundant rDNAs related to the α Proteobacteria. β and γ-Proteobacteria appeared to transiently bloom in the borehole shortly after isolation. Chemical modeling and sulfur isotope analyses of the borehole effluent indicated that microbial sulfate reduction was the major terminal electron-accepting process shortly after isolation, whereas Fe+3 reduction dominated towards the end of the experiment. The persistence of Desulfotomaculum-like bacteria throughout suggests that these organisms adapted to changing geochemical conditions as the redox decreased and pH increased following the isolation of the borehole from the mine atmosphere. The restoration of anaerobic aquatic chemistry to this borehole environment may have allowed microbiota indigenous to the local basalt aquifer to become more dominant among the diverse collection of bacterial lineages present in the borehole. © Taylor & Francis Inc.