Occurrence and distribution of microorganisms in the subsurface of deep-sea hydrothermal vents (sub-vents) were investigated using sub-vent rock samples (cores, approximately 6.5 cm in diameter) collected by the Ocean Drilling Program (ODP). The central parts of the cores (subcores, approximately 1.5 cm in diameter) were extracted in an anaerobic chamber. The degree of contamination by drilling fluid (surface seawater in ODP) was tested using perfluorocarbon (PFC) tracers. The test showed that PFC-traced contamination was limited to core surfaces and was not detected in subcores. Therefore, subcores were used in microbiological analyses for direct counts, adenosine 5-triphoshate (ATP) measurements, thermophilic incubations, and 16S ribosomal RNA gene (16S rDNA) sequences. Microbial cells in the subcores were observed from depths shallower than 97.9 meters below the seafloor (mbsf) by 4' 6'-diamidino-2-phenylindole (DAPI)-epifluorescence microscopy. Similarly, ATP was detected only from depths shallower than 44.8 mbsf by the luciferin-luciferase method. Portions of subcores from various depths, 9.7-301.5 mbsf, were directly incubated anaerobically with a heterotrophic medium at temperatures of 60 degrees C and 90 degrees C. After two weeks, an increase in cell numbers was observed for 60 degrees C-cultures from 59.8-99.4 mbsf samples, and for 90 degrees C-cultures from 69.1-128.9 mbsf samples. The 16S rDNA sequences suggest that microorganisms from the 60 degrees C- and 90 degrees C-cultures are closely related to the thermophilic species belonging to the genera Geobacillus and Deinococcus, respectively. These results indicate 1) existence of microbial habitats in the sub-vent region of subseafloor, and 2) habitat segregation of thermophilic bacteria over the sub-vent thermal gradient.