Nodulation (nod) genes are required for Rhizobium meliloti to invade and stimulate nodule formation in its host, alfalfa. We have established the DNA sequence of nodD, nodA, and nodB, which are part of a gene cluster located 20 kb downstream of nifHDK on the R. meliloti pSym megaplasmid. The nodD open reading frame (308 amino acids) reads from proximal to nifHDK toward distal to nifHDK, divergently from nodA (196 aa) and nodB (217 aa). These two genes read from distal to nifHDK toward proximal, and are just upstream from the previously defined open reading frame for nodC. Fourteen Tn5 insertion sites have been sequenced in nodD, nodA, and nodB, revealing no major hotspots for insertion, but an overall preference for G/C bases at positions 1 and 9 of the 9-bp repeat.