Leguminous plant roots release flavonoids which are sensed by rhizobia and activate genes involved in the development of root nodules during the plant-bacterial colonization process. The key element in this transduction pathway is the product of nodD gene, the NodD protein, a transcriptional activator of the Lys-R family. In the presence of flavonoids, this protein activates the transcription of other nod genes and, usually, represses its own transcription. Herbaspirillum seropedicae is a diazotrophic bacterium found in root tissues of gramineous plants but does not induce nodule formation on the host plant. Its genome was sequenced by the GENOPAR Program, and found to contain a gene similar to nodD. A putative ORF was identified which transcribed in the opposite orientation to the nodD gene whose its predicted product is similar to proteins involved in the degradation of phenolic compounds. Sequence analysis of the intergenic region identified a nod-box and two putative canonical promoter sequences which seem to direct transcription in divergent orientations. This region was cloned upstream of a promoterless lacZ gene in both orientations and β-galactosidase activities were determined to study transcription regulation. We also tested whether the H. seropedicae NodD was able to activate the transcription of nod genes from rhizobia, both in an E. coli overexpressing HsNodD and in H. seropedicae. The nod  genes from rhizobia exhibited very low expression levels both in E. coli and in H. seropedicae suggesting that H. seropedicae NodD interacts weakly with the nod-boxes of these genes. On the other hand, the nodD::lacZ fusion was highly expressed in E. coli and this activity was strongly repressed when HsNodD was overexpressed. This suggests that similar to the nodD of rhizobia, H. seropedicae NodD represses its own transcription. The H. seropedicae nodD gene was cloned in the expression vector pET28a to produce a NodD-His protein which was purified by affinity chromatography on a Hi-Trap chelating column. Gel shift assays showed that the NodD-His protein bound specifically to the nodD gene promoter.