Physiological and biomolecular studies were carried out to characterise five novel mineral-oxidising microorganisms. All were obligately acidophilic, iron-and sulphur-oxidising eubacteria, which grew aerobically or anaerobically (using ferric iron as terminal electron acceptor). They were grouped as: (i) mesophilic Gram-negative bacteria (isolate S-10 and M-12); (ii) mesophilic Gram-positive bacteria (isolate RIV-14 and L-15); and (iii) moderately thermophilic Gram-positive bacteria (isolate GSM). The four mesophilic bacteria were isolated from geothermal sites on the island of Montserrat (W.I) and the moderately thermophile from waste rock from the Golden Sunlight Mine, Montana, U.S.A. A variety of techniques were used to establish the taxonomic relationship of these isolates to known acidophilic, metal-mobilising acidophiles. Isolates S-10 and M-12 shared some morphological and physiological traits in common with Thiobacillus ferrooxidans, though they differed from T ferrooxidans in being able to grow mixotrophically in ferrous iron/yeast extract liquid medium, greater tolerance to hydrogen ions, and in some other areas. In addition, these isolates had a greater (65%) G + C content than the type strain T ferrooxidans (58 - 59%), suggesting that they may represent a novel species. Isolates RIV-14 and L-15 were, in contrast, Gram-positive spore-forming rods. These isolates displayed considerable metabolic flexibility, similar to moderately thermophilic Suljobac;/lus spp.. Comparative analysis of 16S rRNA gene sequences confirmed that RIV-14 and L-15 had a close phylogenetic affiliation with iron-oxidising Su(fobacillus spp.. However, they differed from the latter in being mesophilic and much tolerant to extreme acidity (growth at pH < 1, the lowest values yet recorded for any iron-oxidising prokaryote). Isolate RIV-14 and L-15 differed from each other in some physiological traits, and the binary level comparison of their 16S rRNA gene sequences was low enough (97% sequence identity) to consider them as two novel and distinct species of the Su(fobacillus. Isolate GSM was readily distinguished from the four Montserrat isolates by its thermotolerant nature and its greater capacity for heterotrophic growth. Whilst isolate GSM was found to share some morphological characteristics with the thermotolerant endospore-forrning acidophiles Suljobacillus spp. and Alicyclobacillus spp., biomolecular analysis showed that isolate GSM is phylogenetically distinct from both these and appears to represent a novel bacterial genus. Pure cultures of all five isolates were found to be capable of oxidising pyrite (and the four Montserrat isolates of oxidising chalcopyrite). The Gram-positive Montserrat isolates leached sulphide minerals at much lower pH values and redox potentials than the more well-characterised mineral-oxidising bacteria (T ferrooxidans and Leptospirillum ferrooxidans) suggesting that they may have a useful role in bioleaching of some important mineral ores.