Marine extremely thermophilic methanogens belonging to the genus Methanococcus have been isolated from shallow water systems, deep-sea hydrothermal vents and sea-water-flooded oilfields (Nilsen & Torsvik, 1996; Stetter, 1996; Jeanthon et al., 1998Jeanthon et al., , 1999. Thermophilic methanococci may represent the predominant methanogenic Archaea occurring at deepsea hydrothermal vents (Jones et al., 1989 ; Harmsen et al., 1997), but little is known about their distribution and diversity. One of the reasons for this is the difficulty of rapidly and reliably identifying Methanococcus strains (Boone & Whitman, 1988;Keswani et al., 1996). The paucity of readily identifiable phenotypic characteristics useful for distinguishing methanogens has led to a classification system based largely on phylogenetic assessments (Keswani et al., 1996). 16s rDNA sequence analysis and DNA-DNA hybridization have become standard methods for the investigation of their phylogenetic relationships and for the differentiation among species, respectively (Woese, 1987; Boone & Whitman, 1988;Keswani et al., 1996). However, these laborious and time-consuming methods cannot currently be considered as appropriate tools for routine identification of multiple isolates from the environment. Recently, a simple and rapid approach combining PCR and RFLP analysis has been used successfully for rapid identification at the species level of a variety of organisms including mesophilic methanogens (Vaneechoutte et al., 1992 ;Laguerre et al., 1994;Hiraishi et al., 1995; Blanc et al., 1997; Sat0 et al., 1997;Urakawa et al., 1997).The aim of the present study was to investigate the feasibility of using restriction fragment analysis of PCR-amplified rDNA for the differentiation of the hyperthermophilic species belonging to the genus Methanococcus and for the identification of new isolates from deep-sea hydrothermal vents.Samples of hydrothermal chimneys or sediments were collected in the Guaymas Basin (27" 01' N, 11 1" 24' W) at a depth of 2000 m, on the East Pacific Rise (12" 49' N, 103" 56' W) at a depth of 2600 m and on the Mid-Atlantic Ridge (23" 22' N, 44" 57' W) at a depth of 3500 m. Serial dilutions of hydrothermal samples were carried out in 50 ml vials containing (per litre distilled water): 30 g sea salts (Sigma), 1 g NH4C1, 0.35 g KH,P04, 3.46 g PIPES, 1 g NaHCO,, 2 g Difco yeast extract, 2 g Difco peptone, 1 g sodium acetate, 0-5 g cysteine . HCl, 1 ml trace element mixture (Widdel & Bak, 1992), 30 mg tungstate, 0-5 mg selenate, 1 ml vitamin mixture (Widdel & Bak, 1992), 1 ml thiamin solution (Widdel & Bak, 1992), 0.05 mg vitamin BI2, 1 ml growth-stimulating factors (Pfennig et al., 1981) and 1 mg resazurin. The pH was adjusted to 6-5 using 1 M HCl before autoclaving and the medium was reduced by adding appropriate amounts of sodium sulphide (Na,S. 9H,O). H,/C02 [80/20; 200 kPa (above atmospheric pressure)] was used as the gas phase. Unless indicated otherwise, cultures were incubated at 80 "C for 1-6 d. Pure colonies were isolated by repeated st...