The phylogenetic positions of sulfate-reducing organisms, as revealed from comparisons of small-subunit ribosomal RNA (SSU rRNA), are spread over both the Archaeal and Bacterial domains, though when they evolved is uncertain. The low-branching positions of some of these groups on the Tree of Life have inspired the hypothesis that the metabolic innovation of microbial sulfate reduction is of great antiquity. Only recently, however, have sulfur isotope data from Precambrian rocks begun to emerge that clearly demonstrate sulfate-reducing microbes had evolved by the early Archean. The large spread of δ34S values of microscopic pyrites aligned along growth faces of former gypsum crystals in the ∼3.47-Ga North Pole barite deposit of northwestern Australia provide the oldest evidence of microbial sulfate reduction and the earliest indication of a specific microbial metabolism. The distinct expression of microbial sulfate reduction in this localized and cool sulfate-rich environment provides the oldest date for calibrating the temporal progress of early evolution on the Tree of Life.