Distribution and phylogenetic diversity of microbial communities in hot, deep underground environments in the Hishikari epithermal gold mine, southern part of Kyushu, Japan, were evaluated using molecular phylogenetic analyses. Samples included drilled cores such as andesitic volcanic rock (0.95-1.78 Ma) and the oceanic sedimentary basement rock of Shimanto-Supergroup (100 Ma), as well as geothermal hot aquifer waters directly collected from two different sites: AW-site (71.5 degrees C, pH 6.19) and XW-site (85.0 degrees C, pH 6.80) at a depth of 350 mbls (meters below land surface). Based on PCR-amplified 16S rRNA gene clone analysis, the microbial communities in the drilled cores and the hot aquifer water from the XW-site consisted largely of the 16S rRNA gene sequences, closely related to the sequences often found in marine environments, while the aquifer water from the AW-site contained 16S rRNA gene sequences representing members of Aquificales, thermophilic methanotrophs within the gamma-subdivision of the Proteobacteria and uncultivated strains within the beta-subdivision of Proteobacteria. The cultivable microbial community detected by enrichment cultivation analysis largely matched that detected by the culture-independent molecular analysis.