The bacterial biota of a methanol-fed denitrification reactor used to treat seawater at the Montreal Biodome were investigated using culture-dependent and molecular biology methods. The microbiota extracted from the reactor carriers were cultivated on three media. Three isolate types were recovered and their 16S ribosomal DNA (rDNA) genes were determined. The analysis showed that the isolate types were related to alpha-Proteobacteria. They are members of the Hyphomicrobium and Paracoccus genera and the Phyllobacteriaceae family. Uncultured bacteria were identified through a 16S rDNA library generated from total DNA extracted from the microbiota. Clones were screened for different restriction profiles and for different DGGE (denaturing gradient gel electrophoresis) migration profiles. More than 70% of clones have the same restriction profile, and the sequence of representative clones showed a relation with the Methylophaga members of the Piscirickettsia family (gamma-Proteobacteria). Sequences from other profiles were related to bacterial species involved in denitrification. The number of species in the denitrification reactor was estimated at 15. Bacterial colonization on newly added carriers in the denitrification reactor was monitored by PCR-DGGE. The DGGE migration profiles evolved during the first 5 weeks and then remained essentially unchanged. PCR-DGGE was also used to monitor the microbial profiles in various aquarium locations. As expected, bacterial populations differed from one location to another, except for the sand and trickling filters which presented similar DGGE migration profiles.