16S ribosomal (r) DNA from 9 members of the genus Azospirillum and the type strain of Aquaspirillum itersonii (NCIMB 9070) was amplified using the PCR. Of the strains received as Azospirillum, five were the type strains for the validly described species whilst the remaining 4 strains had been deposited as ''Azospirillum'' sp. The amplified rDNA was directly sequenced using the linear PCR reaction and the sequences analyzed using a distance matrix method to determine the intergeneric and intrageneric relationships of Azospirillum, All of the type strains of Azospirillum, together with 2 of the unassigned strains, formed a deep branching clade within the alpha-proteobacteria in which Azospirillum amazonense, A. brasilense, A. halopraeferens, A. irakense and A. lipoferum constituted phylogenetically well defined species. A. brasilense, A. lipoferum and A. halopraeferens were more closely related to each other than to A. amazonense or A. irakense. Two of the unassigned strains (DSM 4834, DSM 4835) were closely related to, but distinct from, A. irakense. The other two ''Azospirillum'' sp. (DSM 1726, 1727) were misclassified. ''Azospirillum'' sp. DSM 1726 is closely related to Agrobacterium tumefaciens while DSM 1727 was recovered as a deep branching lineage within the gamma-proteobacteria. The anoxygenic phototroph Rhodospirillum centenum was recoverd within the radiation defined by Azospirillum an close to A. irakense. The nearest relatives to the azospirilla were Magnetospirillum magnetotacticum and Rhodospirillum fulvum. Aquaspirillum itersonii formed a distinct lineage within the alpha-proteobacteria but its exact position was unstable and influenced by the composition of the alignment.