Four substitutions at serine residue 160 which increase the activity of the σ54-dependent activator protein NTRC in the absence of NTRB have been analysed in detail. Mutagenesis of the putative phosphoacceptor site of NTRC and analysis of double mutants indicate that the positive control function of the S160W and S160C mutants is phosphorylation-dependent, whereas the activity of the S160Y and S160F mutants is phosphorylation-independent. This was confirmed with two purified mutant proteins in vitro. Occupancy of tandem NTRC-binding sites upstream of the Klebsiella pneumoniae nifL promoter by S160W protein is also phosphorylation-dependent in contrast to occupancy by S160F protein, confirming that both the DNA-binding and activator functions of NTRC are influenced by phosphorylation. The S160W and S160C mutants are apparently more responsive than wild-type protein to ‘cross-talk’ by other members of the histidine protein kinase family but are less responsive to phosphorylation and dephosphorylation mediated by NTRB.