The phylogeny and taxonomy of the mesophilic methane-producing archaea of the order Methanococcales were examined by DNA relatedness, 16s rRNA sequence analysis, cellular protein pattern, and phenotypic methods. The mesophilic species Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltaei, and 44Methanococcus aeolicus" formed a deep group with 5 to 30% DNA relatedness and 92 to 96% 16s rRNA sequence similarity. Twenty-two additional isolates and Methanococcus deltae were similar to the type strain of either M. voltaei or M. maripaludis. Two isolates, strains A2 and A3, exhibited 37% DNA relatedness and 99.2% 16s rRNA sequence similarity to M. voltaei PST (T = type strain). In the absence of phenotypic differences, these organisms were assigned to M. voltaei. Similarly, four autotrophic isolates, strains C5, C6, C7, and C8, exhibited 54 to 69% DNA relatedness and 99.2% 16s rRNA sequence similarity to M. maripaludis JJT and were assigned to M. maripaludis. While these isolates were sufficiently genetically diverse to justify classification in novel species, few differences were apparent in the phenotypic properties available for measurement. Thus, the phenotypic properties of these lithotrophic archaea were highly conserved and poor indicators of genetic diversity. Partial sequencing of about 200 bases of both the 16s and 23s rRNAs of the isolates demonstrated allelic diversity within methanococcal species. This allelic diversity did not correlate with diversity measured by DNA relatedness, cellular protein pattern, and other methods. Similarly, antisera to whole cells of the type strains did not cross-react strongly to whole cells of strains that were genetically similar, and serological cross-reactivity was not a useful taxonomic method for methanococci. Lastly, on the basis of the results of 16s rRNA sequence analyses and biochemical data, the ancestor of the mesophilic methanococci may have been an autotrophic thermophile.The genus Methanococcus belongs to the family Methanococcaceae and the order Methanococcales, which is one of five orders within the Archaea that include methanogenic bacteria (3). The order Methanococcales is composed of coccoid methane-producing bacteria of marine origin. Distinctive features of this group include rapid growth at either mesophilic or thermophilic temperatures, cell envelopes composed of protein S-layers, and a nutritional requirement for selenium (6,18,23,44,45). To date, eight Methanococcales species belonging to two families and four genera have been described. Five species, Methanococcus vannielii, Methanococcus voltaei, Methanococcus maripaludis, Methanococcus deltae, and "Methanococcus aeolicus," are mesophilic, and three species, Methanococcus ("Methanothermococcus") thermolithotrophicus, Methanococcus ("Methanoignis") igneus, and Methanococcus ("Methanocaldococcus~7) jannaschii (4) (proposed genus names in parentheses), are thermophilic. The relationships of all of the mesophilic species except "M. aeolicus" have been determined by 16s rRNA catalog...