Two species belonging to a novel genus of archaea, designated Picrophilus oshimae and Picrophilus torridus, have been isolated from two different solfataric locations in northern Japan. One habitat harboring both organisms was a dry, extremely acidic soil (pH < 0.5) that was heated by solfataric gases to about 55؇C. In the laboratory both species grew heterotrophically on yeast extract and poorly on tryptone under aerobic conditions at temperatures between 45 and 65؇C; they grew optimally at 60؇C. The pH optimum was 0.7, but growth occurred even around pH 0. Under optimal conditions, the generation time was about 6 h, yielding densities of up to 10 10 cells per ml. The cells were surrounded by a highly filigreed regular tetragonal S-layer, and the core lipids of the membrane were mainly bis-phytanyltetraethers. The 16S rRNA sequences of the two species were about 3% different. The complete 16S rRNA sequence of P. oshimae was 9.3% different from that of the closest relative, Thermoplasma acidophilum. The morphology and physiological properties of the two species characterize Picrophilus as a a novel genus that is a member of a novel family within the order Thermoplasmales.Thermoacidophilic microorganisms thrive on coal refuse piles and in the upper oxygenic zones of acidic geothermally heated waters in solfataras. Sulfuric acid arises in these habitats from the oxidation of H 2 S, either spontaneously or by the action of sulfur-oxidizing bacteria (1). Except for some heterotrophic species of the bacterial genus Bacillus, all organisms isolated from acidic environments with temperatures above 50ЊC have been archaea of the orders Sulfolobales and Thermoplasmales (8, 9). Members of the Sulfolobales are extreme thermophiles that grow optimally at between 75 and 85ЊC. With one exception, they are obligate or facultative heterotrophic aerobes and/or chemolithoautotrophs; in the latter case they are capable of gaining energy by oxidation of sulfur compounds to sulfuric acid or by reduction of sulfur to H 2 S (20). The first described representative of the order Thermoplasmales, Thermoplasma acidophilum, was isolated by Darland et al. (2) from a coal refuse pile. It is unique among the archaea in being devoid of a cell envelope. T. acidophilum is a facultatively anaerobic heterotroph that requires complex organic extracts for growth. Under anerobic conditions, it grows by sulfur respiration (19,21). T. acidophilum grows optimally at 59ЊC and was the prokaryotic record-holder in acidophily, with a pH optimum of around 1.8 to 2 and the ability to still, although barely, grow at around pH 0.4 (1, 2). T. acidophilum and related acidophiles with similar morphologies and physiological properties that have been combined in the taxon Thermoplasma volcanium were later found in various natural habitats around the world (11, 18).Here we report on the isolation and characterization of members of two species of a novel genus and family of thermoacidophilic archaea, which have been obtained by sampling solfataric environments in north...