Enrichments for anaerobic, organotrophic hyperthermophiles were performed with hydrothermal chimney samples collected from the Mid-Atlantic Ridge at a depth of 3,550 m (23degrees22'N, 44degrees57'W) and the Guaymas Basin (27degrees01'N, 111degrees24'W) at a depth of 2,616 m. Positive enrichments were submitted to gamma-irradiation at doses of 20 and 30 kGy. Two hyperthermophilic, anaerobic, sulfur-metabolizing archaea were isolated. Strain EJ1(T) was isolated from chimney samples collected from the Mid-Atlantic Ridge after gamma-irradiation at 20 kGy, and strain EJ2(T) was isolated from the Guaymas Basin after gamma-irradiation at 30 kGy. Only strain EJ2(T) was motile, and both formed regular cocci. These new strains grew between 55 and 95 degreesC with the optimal temperature being 88 degreesC. The optimal pH for growth was 6.0, and the optimal NaCl concentration for growth was around 20 g l(-1). These strains were obligate anaerobic heterotrophs that utilized yeast extract, tryptone, and peptone as a carbon source for growth. Ten amino acids were essential for the growth of strain EJ1(T), such as arginine, aspartic acid, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tyrosine, and valine, while strain EJ2(T) was unable to grow on a mixture of amino acids. Elemental sulfur or cystine was required for EJ2(T) growth and was reduced to hydrogen sulfide. Rifampicin inhibited growth for both strains EJ1(T) and EJ2(T). The G+C contents of the genomic DNA were 52.3 and 54.5 mol% for EJ1(T) and EJ2(T), respectively. As determined by 16S rRNA gene sequence analysis, these strains were more closely related to Thermococcus gorgonarius, T. celer, T. guaymasensis, T. profundus, and T. hydrothermalis. However, no significant homology was observed between them with DNA-DNA hybridization. These novel organisms also possess phenotypic traits that differ from those of its closest phylogenetic relatives. Therefore, it is proposed that these isolates, which are amongst the most radioresistant hyperthermophilic archaea known to date with T. gammatolerans (Jolivet et al. 2003a), should be described as novel species T. marinus sp. nov. and T. radiotolerans sp. nov. The type strain of T. marinus is strain EJ1(T) (=DSM 15227(T)=JCM 11825(T)) and the type strain of T. radiotolerans is strain EJ2(T) (=DSM 15228(T)=JCM 11826(T)).