Specific molecular determination and classification of ammonia-oxidizing bacteria have relied on the use of conventional markers such as 16S rDNA. However, this gene does not satisfactorily provide a wide vision of all phylogenetic lineages. Despite the initial expectations, the use of functional genes as for example amoA has only been useful to corroborate the established taxonomy. Ammonia-oxidizing bacteria constitute a physiological group that crosses over principal phylogenetic radiations. Therefore, it is necessary to look for novel functional markers, which are needed for both diversity and taxonomic studies. In this work, the available amoB sequences have been used to design a new degenerate set of primers flanking a ca. 500-bp region. Partial amoB gene sequences of up to 16 AOB strains (5 Nitrosomonas, 10 Nitrosospira, and 1 Nitrosococcus) belonging to both the beta- and the gamma-Proteobacteria have been obtained. Comparison of both DNA and deduced amino acid sequences results in three subgroups, two of them of the beta-Proteobacteria and a third one of the gamma-Proteobacteria displaying 75% and 35% homology in their deduced amino acid sequences, respectively. This gene has proven to be a suitable molecular marker to study AOB, as well as providing a new insight into the classification of this group.