A moderately thermophilic mixed culture, MT, and the thermophilic Sulfolobus acidocaldarius strain BC were studied for their response to arsenic in a defined medium and also in media containing a pyrite and an arsenical pyrite flotation concentrate. In defined medium, the individual constituents of the MT culture exhibited a high tolerance to arsenite and arsenate compared to S. acidocaldarius strain BC. When grown on increasing concentrations of the pyrite flotation concentrate, both cultures had similar specific leaching rates over the various concentrations of the mineral substrate. In contrast, S. acidocaldarius strain BC exhibited a decreasing specific teaching rate when grown on the arsenical pyrite while the MT culture was not affected. In addition, arsenic added to cultures of S. acidocaldarius strain BC growing with pyrite as a growth substrate inhibited further growth, while added arsenic had no effect on the MT culture growing on the pyrite. These data indicate that the moderately thermophilic, arsenic-resistant MT culture was able to leach arsenical pyrite more efficiently than was the S. acidocaldarius strain BC culture at high concentrations of the mineral. This emphasizes the fact that proper culture selection is an important parameter when developing commercial processes involving arsenic-containing minerals.