Fluorescent in situ hybridization (FISH) method with 16S rRNA-targeted oligonucleotide probes was used for quantitative estimation of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in a Johkasou. Although the occupation ratios of AOB and NOB increased as nitrification progressed, about one month later, the occupation ratios decreased, despite showing good nitrification ability. Furthermore, even when urea was added to the feeding wastewater to raise the amount of T-N, the occupation ratios of both nitrifying bacteria remained constant. For further investigation, denaturing gradient gel electrophoresis (DGGE) was used to study the community structure of AOB in the Johkasou. As a result, DGGE band patterns and following sequence analysis revealed that the community structure of AOB was complicated and changed during this experiment. It was suggested that even if the occupation ratio of AOB to eubacteria was constant, the majorities of AOB were changed through temperature and load fluctuation. The combination of FISH and PCR-DGGE provides new information that was not available by conventional cultivation-based methods.