Examination of nolA revealed that NolA can be uniquely translated from three ATG start codons. Translation from the first ATG (ATG1) predicts a protein (NolA1) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins. Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA2 and NolA3, respectively, lacking the DNA-binding domain. Consistent with this, immunoblot analyses of Bradyrhizobium japonicum extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of nolA from the three ATG initiation sites. Site-directed mutagenesis was used to produce derivatives of nolA in which ATG start sites were sequentially deleted. Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed. Translational fusions of the nolA mutants to a promoterless lacZ yielded functional fusion proteins in both Escherichia coli and B. japonicum. Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the B. japonicum nod genes. The expression of both NolA2 and NolA3 requires the presence of NolA1. NolA1 or NolA3 is required for the genotype-specific nodulation of soybean genotype PI 377578.