Starting with plasmid pSA30 which contains an intact nifHDKY operon, we selected mutants that no longer inhibited nitrogen fixation in Klebsiella pneumoniae. Three categories of mutants were found among eight mutant plasmids examined in detail. Three mutant plasmids carried a single-base-pair (bp) change at position -12 or -14 relative to the nifH transcription start site. These mutations were located in a previously described consensus sequence found in the -10 to -15 region of nif promoters. Four of the mutant plasmids contain lesions considerably upstream from the start point of transcription. Two of these upstream mutations are identical 112-bp deletions of nucleotides -72 to -184, and two others are a single-bp change at position -136. The final plasmid does not contain a mutation within a 525-bp region which includes 289 bp upstream from the nifH ATG initiation codon and which extends 69 codons into the nifH gene. All eight of the mutant pSA30 plasmids failed to complement a chromosomal nifH mutation, suggesting that the mutations that block inhibition of nif expression also prevent transcription of the nifHDKY operon.