The eubacterial enhancer-binding proteins activate transcription by binding to distant sites and, simultaneously, contacting the RNA polymerase σ54 promoter complex (Eσ54). The positive control function is located at the central domain of these proteins, but it is not know which specific region has the determinants for the interaction with Eσ54. Here, we present genetic evidence that a small region of hydrophobic amino acids, previously denominated C3, at the central domain of Bradyrhizobium japonicum NifA is involved in positive control. We obtained 26 missense mutants along this conserved region. Among these, only strains expressing the NifAF307→Y and NifAA310→S mutant proteins retained some of the transcriptional activity (< 20%), whereas those carrying NifAE298→D and NifAT308→S had very low but detectable activity (< 1.0%). The rest of the NifA mutants did not induce any measurable transcriptional activity. When expressed in the presence of wild-type NifA, the great majority of the mutants displayed a dominant phenotype, suggesting that their oligomerization determinants were not altered. In vivo dimethyl-sulphate footprinting experiments for a subset of the NifA mutants showed that they were still able to bind specifically to DNA. Analysis of intragenic supressors highlight the functional role of a hydroxyl group at position 308 to activate transcription.