The Rhizobium meliloti nodD gene products are positive transcriptional activators of genes required for early stages of nodule morphogenesis in the R. meliloti-alfalfa symbiosis (nod genes). The regulatory activity of NodD, a member of the LysR family of activator proteins, is mediated in part through its binding to conserved DNA sequences termed nod boxes which lie upstream of the inducible nod genes. Here we use interference footprinting to identify two NodD binding sites in the nodA, nodF and nodH nod boxes. These two binding sites are located on the same face of the DNA helix and can be separated by an additional 10 bp with retention of activity. By systematic alteration of the phasing of the two binding sites on the DNA helix, we showed that only constructs which contain both sites on the same side of the helix are recognized by NodD as determined by migration retardation assay and by in vivo activation of nod box-lacZ fusions. Moreover, NodD apparently induces a bend in the DNA upon binding at the nod box as shown by migration retardation behavior of circularly permuted nod box fragments.