Four formate-utilizing methanogens were isolated from ovine (strain KM1H5-1P(T)) and bovine (strains AK-87, OCP and ZA-10(T)) rumen contents. Based on 16S rRNA gene sequence analysis, the methanogen strains were found to belong to the order Methanobacteriales in the genus Methanobrevibacter. Strains ZA-10(T) and KM1H5-1P(T) gained energy for growth by the reduction of CO(2) to CH(4) using H(2) or formate exclusively as electron donors. Increasing formate concentrations to 220 mM in batch cultures increased the growth of strain KM1H5-1P(T) but did not affect the growth of strain ZA-10(T). Substrate specificity and resistance to cell-wall lysis supported the affiliation of the strains to the genus Methanobrevibacter. Strains ZA-10(T) and KM1H5-1P(T) showed 16S rRNA gene sequence similarity of 98.0 and 98.6 % to their closest recognized relatives, Methanobrevibacter thaueri CW(T) and Methanobrevibacter ruminantium M1(T), respectively. DNA-DNA hybridization experiments indicated that the strains were not affiliated at the species level to their closest recognized relatives, with DNA reassociation values of only 28 % between strains ZA-10(T) and Methanobrevibacter thaueri CW(T) and <25 % between strains KM1H5-1P(T) and Methanobrevibacter ruminantium M1(T). Based on the data presented, the new strains are considered to represent two novel species of the genus Methanobrevibacter, for which the names Methanobrevibacter millerae sp. nov. (type strain ZA-10(T)=DSM 16643(T)=OCM 820(T)) and Methanobrevibacter olleyae sp. nov. (type strain KM1H5-1P(T)=DSM 16632(T)=OCM 841(T)) are proposed.