The genetic analysis of genes from prokaryotic species for which experimental genetic systems have not yet been developed is often limited by the difficulty of producing mutations in those genes. We report here a general technique applicable to Gram-negative prokaryotes for site-directed mutagenesis of cloned DNA fragments which we have applied to the study of the symbiotic nitrogen fixation genes of Rhizobium meliloti. In particular, we mutagenized cloned R. meliloti restriction fragments in Escherichia coli with transposon Tn5 and then replaced the wild-type parental DNA sequences with the mutant DNA sequences in the R. meliloti genome. Using this method we show that an R. meliloti DNA restriction fragment, cloned previously on the basis of homology to Klebsiella pneumoniae nif genes, contains gene(s) essential for symbiotic nitrogen fixation. In addition, we use this method to construct a physical genetic map of a subset of the R. meliloti nif genes.