The diversity of Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum tritici and Ochrobactrum grignonense in agricultural soil and on the wheat rhizoplane was investigated. O. anthropi was isolated both from soil and from the rhizoplane, O. intermedium and grignonense only from bulk soil, and O. tritici only from the wheat rhizoplane. On the genetic level, the immunotrapped isolates and a number of strains from culture collection mainly of clinical origin were compared with rep-PCR profiling using BOX primers, and a subset of these isolates and strains using REP primers. The isolates clustered according to their species affiliation. There was no correlation between rep clusters of O. anthropi isolates and habitat (place of isolation). The genetic diversity of Ochrobactrum at the species level as well as microdiversity of O. anthropi (number of BOX groups) was higher in soil than on the rhizoplane. Similarity values from genetic rep-PCR profiles correlated positively with DNA-DNA reassociation percentages. Isolates with >80.7% similarity in BOX profile and >86.4% in rep profile clustered within the same species. Similarity analysis of rep-PCR profiles is hence an alternative to DNA-DNA hybridization as a genomic criterion for species delineation within the genus Ochrobactrum. We used the substrate utilization system BIOLOG-GN to compare the immunotrapped isolates on the phenetic level. For the isolates from bulk soil, substrate utilization versatility (number of utilized substrates) and substrate utilization capacity (mean conversion rate of substrates) were slightly but significantly higher than for the isolates from the rhizoplane. This trend was also seen using API 20E and 20NE systems. Plate counts of total bacteria and the number of immunotrapped Ochrobactrum isolates per gram dry weight were higher for the rhizoplane than for the soil samples. The results of genetic and phenotypic analyses indicated a 'rhizosphere effect'; the diversity and metabolic capacity of Ochrobactrum isolates were higher in bulk soil, and the population density was higher on the wheat rhizoplane.