Rhizobium meliloti strains carrying either of two overlapping deletions (delta 5408 and delta F114) of the megaplasmid pRmeSU47b form nodules on alfalfa which fail to fix N2 (Fix-). Strains carrying these deletions also fail to fluoresce on media containing calcofluor, indicating a defect in synthesis of the acidic exopolysaccharide (Exo-) of R. meliloti. We have isolated cosmid clones (pTH21 and pTH22) which complement the Fix- but not the Exo- phenotype of the strains carrying the delta 5408 and delta F114 deletions. In addition, cosmid clones which complement the Exo- phenotype fail to complement the Fix- phenotype of these deletions; thus, the Exo- phenotype is not related to the Fix- phenotype. A 5-kb region within a 7.3-kb BamHI restriction fragment was found to be required for complementation of the Fix- phenotype of the delta 5408 and delta F114 deletion strains. Tn5 insertions in the 5-kb region generated a Fix- phenotype when recombined into the wild-type genome. We have designated this locus ndvF, for nodule development. TnphoA mutagenesis of this region generated active alkaline-phosphatase gene fusions, indicating that ndvF encodes extracytoplasmic protein(s). Induction of nodules by the ndvF mutants was delayed by 2 to 3 days compared with induction by the wild-type strain. Light microscopy of nodules elicited by strains carrying the large 150-kb delta F114 deletion, a 12-kb deletion removing ndvF, or an individual ndvF::Tn5 insertion mutation demonstrated that many nodules contained few infected cortical cells, indicating that nodule development was blocked early in the infection process, before the release of bacteria from the infection threads.