The mutation nhaAup (antup) has now been identified as a Glu134 to Ala substitution in NhaR and designated nhaR1. This was demonstrated by sequence analysis showing that the mutant contains a wild-type nhaA but nhaR1 instead of nhaR and by the finding that nhaR1 cloned in a plasmid confers the NhaAup phenotype. Na+ (107 mM) increases by 5- to 10-fold the level of nhaA transcripts, similar to the effect on the NhaR-mediated expression of a nhaA'-'lacZ fusion. These results are in agreement with the notion that nhaR is a positive regulator which controls Na(+)-dependent transcription of nhaA. The promoter region of nhaR and nhaR1 was found to reside within the BglII-BamHI fragment of the C-terminal sequences of nhaA. The mutation nhaR1, while increasing dramatically the level of transcription, reduces the requirement for Na+ by 3- to 5-fold both for nhaA transcription and for the nhaR1-mediated expression of nhaA'-'lacZ fusion. NhaR1, like NhaR, binds specifically to the promoter region of nhaA. However, at equal protein concentration NhaR1 binds more DNA and the NhaR1-DNA complex shows higher mobility than that of NhaR-DNA, suggesting the existence of two different binding complexes. Yet in this assay the DNA binding pattern of neither NhaR nor NhaR1 was affected by the addition of Na+. The possible relevance of these two DNA-binding complexes to the Na(+)-induced NhaR-mediated expression is discussed.