The enrichment and characterization of anaerobic ammonium-oxidizing biofilm cultures are ongoing in our laboratories. Biomass, with a predominately red color, demonstrating simultaneous removal of ammonium and nitrite under autotrophic and anoxic conditions, which is characteristic of anaerobic ammonium-oxidizing planctomycetes, was enriched and maintained for an extended period on a polyester nonwoven carrier. To investigate the bacterial composition of the mature biofilm community, 16S rDNA sequences were amplified by PCR and comparative analyses using DNA databases were conducted. Only one sequence had a notable similarity (92.2%) to that of the first discovered anaerobic ammonium-oxidizing planctomycete and lesser, yet significant, similarities to the 16S rDNA sequences of other recently reported anaerobic ammonium-oxidizing strains. The newly discovered strain (designated KSU-1) reported here was dominant among detectable members of the biofilm community. By fluorescence imaging, KSU-1 was shown to form spherical clusters wrapped in a thin layer of Zoogloea sp. Possible interactions and interdependencies of these two species are discussed with regard to the putative unculturability of the anaerobic ammonium-oxidizing planctomycetes.