Despite recent advances in commercially optimized identification systems, bacterial identification remains a challenging task in many routine microbiological laboratories, especially in situations where taxonomically novel isolates are involved. The 16S rRNA gene has been used extensively for this task when coupled with a well-curated database, such as EzTaxon, containing sequences of type strains of prokaryotic species with validly published names. Although the EzTaxon database has been widely used for routine identification of prokaryotic isolates, sequences from uncultured prokaryotes have not been considered. Here, the next generation database, named EzTaxon-e, is formally introduced. This new database covers not only species within the formal nomenclatural system but also phylotypes that may represent species in nature. In addition to an identification function based on Basic Local Alignment Search Tool (blast) searches and pairwise global sequence alignments, a new objective method of assessing the degree of completeness in sequencing is proposed. All sequences that are held in the EzTaxon-e database have been subjected to phylogenetic analysis and this has resulted in a complete hierarchical classification system. It is concluded that the EzTaxon-e database provides a useful taxonomic backbone for the identification of cultured and uncultured prokaryotes and offers a valuable means of communication among microbiologists who routinely encounter taxonomically novel isolates. The database and its analytical functions can be found at http://eztaxon-e.ezbiocloud.net/.