Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.Archaea can thrive in various natural and engineered environments, and many of them can grow in habitats at the extreme limits (in terms of temperature, pH, salinity, anaerobiosis, etc.) of conditions that allow life on earth (7a). From a pragmatic perspective, archaeal extremophiles can be exploited for biotechnological applications (9a, 25a), while methanogens mediate the terminal step of the carbon cycle (through methanogenesis) in anaerobic environments, including rumens and anaerobic digestors (2b, 20a). The methanogens present in the latter two habitats have recently received renewed interest because it has been recognized that ruminal methanogens contribute significantly to emission of methane (a potent greenhouse gas) and to loss of feed energy (22a), while the methanogens present in anaerobic digestors produce methane biogas as a valuable bioenergy source from a variety of types of biomass (2a). Since Archaea are typically fastidious organisms that are difficult to culture in the laboratory, they are often analyzed by molecular biology techniques (1, 28).Community profiling by PCR-denaturing gradient gel electrophoresis (DGGE) has been widely used to examine both temporal succession in microbial communities and spatial variations in microbial diversity in the same type of microbial communities (21, 24). It is especially useful for identifying samples of interest for in-depth analyses in a large number of samples (9,13,16,20). However, even using universal primers, Archaea were probably not accounted for in such studies because sequencing of excised DGGE bands rarely yielded Archaea-like sequences (13,14). This is probably attributable to the low abundance of Archaea in most microbial communities and/or primer mismatch. Thus, Archaea-targeting PCR-DGGE has been used increasingly for profiling Archaea in various environmental samples (1,17,27,33). A recent study also reported profiling of ruminal methanogens by PCR-DGGE, but no primer was reported (8). In all these studies different primer sets were used and different hypervariable (V) regions were targeted, but no justification or rationale was presented. In a previous study we noted that different V regions of the bacterial 16S rRNA gene (rrs) can produce different DGGE patterns (number and separation of DGGE bands) (31). We hypothesized that this is also true for Archaea-specific PCR-DGGE. In this study we first evaluated a battery...