Prosthecobacter fusiformis is morphologically similar to caulobacters; however, it lacks a dimorphic life cycle. To determine the relatedness of the genus Prosthecobacter to dimorphic caulobacters and other prosthecate members of the alpha subgroup of the Proteobacteria (alpha-Proteobacteria), we isolated and sequenced 16S rRNA genes from four Prosthecobacter strains. Surprisingly, the results of phylogenetic analyses placed the fusiform caulobacters in a deeply rooted division of the Bacteria that was most closely affiliated with the Planctomyces-Chlamydia group and only distantly related to the alpha-Proteobacteria. The genus Prosthecobacter shares a common lineage in this division with Verrucomicrobium spinosum, a polyprosthecate, heterotrophic bacterium. Consistent with this phylogenetic placement, menaquinones were isolated from Prosthecobacter strains and menaquinones have been isolated from Verrucomicrobium strains and planctomycetes but not from members of the alpha-Proteobacteria. Thus, the genus Prosthecobacter is a second genus in the recently described order Verrucomicrobiales. Members of the genus Prosthecobacter are susceptible to beta-lactam antibiotics and contain mesodiaminopimelic acid, indicating that they, unlike members of the Planctomycetales or Chlamydiales, have peptidoglycan cell walls. This major phenotypic difference, together with the phylogenetic independence of the verrucomicrobia, indicates that these bacteria and the sources of related 16S ribosomal DNAs obtained from soils, freshwater, and the marine pelagic environment represent an unrecognized division of the Bacteria.