ABSTRACT The transcriptional activation of the NifA-dependent σ ⁵⁴ promoter of the Rhizobium leguminosarum hydrogenase structural genes hupSL (P ₁ ) has been studied through gel retardation analysis and detailed mutagenesis. Gel retardation analysis indicated the existence of a physical interaction between NifA and the promoter. Extensive mutagenesis followed by in vivo expression analysis showed that three sequences of 4 bases each (−170 ACAA −167, −161 ACAA −158, and −145 TTGT −142) are required for maximal stimulation of in vivo transcription of the P ₁ promoter. The arrangement of these upstream activating sequences (ACAA N ₅ ACAA N ₁₂ TTGT) differs from the canonical 5′ACA N ₁₀ TGT 3′ UAS structure involved in NifA-dependent activation of nif/fix genes. Mutant promoter analysis indicated that the relative contribution of each of these sequences to P ₁ promoter activity increases with its proximity to the transcription start site. Analysis of double mutants altered in two out of the three enhancer sequences suggests that each of these sequences functions in NifA-dependent activation of the P ₁ promoter in an independent but cooperative mode. The similarities and differences between cis elements of hup and nif/fix promoters suggest that the structure of the P ₁ promoter has adapted to activation by NifA in order to coexpress hydrogenase and nitrogenase activities in legume nodules.