Cultivation-based and molecular approaches were used to characterize the phylogenetic composition and structure of the microbial community in an extremely acidic (pH 2.0) acid mine drainage (AMD) associated with Pb/Zn mine tailings that were undergoing vigorous acid generation. Acidophilic bacteria were isolated and enumerated on solid media, and were found to be restricted to isolates related to Acidithiobacillus ferrooxidans and Acidiphilium cryptum. By contrast, cloning and phylogenetic analysis of 16S rRNA genes revealed that, although low in total taxonomically distinct groups, the tailings AMD ecosystem harbored a wide range of phylogenetically diverse microbes. Of the 141 clones examined, 104 were phylogenetically affiliated with the recently discovered, iron-oxidizing Leptospirillum group III within the Nitrospira. It thus appears that iron serves as the major electron donor in this habitat. Thirty clones were affiliated with the Proteobacteria, half of which belonged to organisms related to Alphaproteobacteria species capable of ferric iron reduction. Other clones were grouped with Betaproteobacteria and Gammaproteobacteria (six clones each), and even with Deltaproteobacteria (three clones), a subdivision with anaerobic sulfate or metal (iron) reduction as the predominant physiological trait of its members. Finally, four clones were clustered within the Firmicutes and the Acidobacteria. Approximately half of the sequence types representing the majority of the total clones fell into lineages that are poorly represented by cultured organisms or have thus far been represented by only a few environmental sequences. Thus, the present study extends our knowledge of the biodiversity of microorganisms populating highly acidic AMD environments.