An 18 kb region adjacent to and surrounding the genes for nitrogenase (nif) was cloned from the genome of the symbiotic nitrogen-fixing species Rhizobium meliloti. A total of 31 Tn5 insertions in the nif region were constructed and assayed for their effect on symbiotic nitrogen fixation (Fix phenotype). Fix- insertions were found in two clusters, one 6.3 kb region not containing essential symbiotic genes. The locations of at least three transcription units containing Fix genes were deduced from complementation analysis between genomic nif::Tn5 insertions and nif::Tn5 insertions on mobilizable cloning vectors. The locations of R. meliloti genes nifH, nifD and nifK, which code for the single subunit of the nitrogenase Fe protein and for the two subunits of the nitrogenase MoFe protein respectively, were determined by DNA hybridization to cloned Klebsiella pneumoniae nif genes and by comparison of partial R. meliloti DNA sequences with K. pneumoniae nif gene sequences. R. meliloti nifH, D and K are located in the 6.3 kb fix-::Tn5 cluster and are transcribed in the order nifH, nifD, nifK, which is the same order as in K. pneumoniae.