Monoclonal antibody method is very useful and helpful for in situ detection of the specific bacteria like nitrifiers in a mixed bacterial habitat such as a biofilm. In this study, the specificities of monoclonal antibodies raised against Nitrosomonas europaea (IFO14298) and Nitrobacter winogradskyi (IFO14297) were checked by sandwich ELISA. As a result, it was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria and specificity for various genus and species of nitrifying bacteria. The spatial distribution of N. europaea and N. winogradskyi immobilized by entrapped-type and attached-type PEG gel pellet introduced into the nitrification reactor was examined by in situ fluorescent antibody technique. As a result, it was found that N. winogradskyi was more sensitive to the damage during entrapping process than N. europaea. Whereas N. winogradskyi existed only near the surface area, N. europaea existed near the surface area and at inner part of gel pellet. On the other hand, in attached-type PEG gel pellet, the high density areas of N. winogradskyi were observed compared with entrapped-type PEG gel pellet. It was suggested that this fluorescent antibody technique was utilized for understanding the microbial community of nitrifying bacteria as a quick and favorable tool.