The prokaryotic communities in deep subseafloor sediment collected during Ocean Drilling Program (ODP) Leg 204 from the South Hydrate Ridge (SHR) on the Cascadia Margin were analyzed by 16S rRNA gene clone sequencing and a fluorescent quantitative PCR technique. The microbial communities came from sites with contrasting geological characteristics on the SHR: sites 1244 and 1245 (located on the flank of the ridge, hydrate-rich sediment) and site 1251 (located on the slope basin of SHR, hydrate-poor sediment). The overall copy numbers of the 16S rRNA gene, and the proportion of archaeal 16S rRNA gene in all 16S rRNA gene community in sediment were larger on the slope basin than on the flank of the SHR. Archaeal community structure around the sulfate-methane transition zone at site 1251 (4.5 mbsf) was intensively investigated using two different PCR primer sets. A relatively abundant distribution of the 16S rRNA gene sequences related to mesophilic methanogen of the genus Methanoculleus was identified at a depth of 43.2 mbsf, and suggested that the methanogens occur in relatively shallow zones of sediment. This study demonstrated that the subseafloor microbial communities shown by 16S rRNA gene clone analyses were not directly associated with subseafloor methane hydrate deposits.