Previous analysis of B. japonicum nifH'- and nifD'-'lacZ translational fusions showed that these promoters could be activated by the K. pneumoniae nifA plus the E. coli ntrA gene products. To study the functions of the DNA 5' to these promoters, plasmids carrying deletions in this region were constructed and analyzed in vivo in a heterologous system consisting of an E. coli (NtrA+) background with a plasmid that constitutively expresses the K. pneumoniae nifA gene. Activation of the B. japonicum promoters was completely dependent on sequences located between positions -165 and -100, relative to the start of transcription. Some of the nifD deletion-fusions were mobilized to the wild-type B. japonicum and the exconjugants tested in an ex planta micro-aerobic system, and also used to infect soybean seedlings. The time course of derepression was followed by assaying beta-galactosidase activity from samples withdrawn from the microaerobic cultures or from root-nodule extracts. The results conclusively show that in the homologous system the sequences upstream of the promoter are required to achieve wild-type activity.