Mutants of R. capsulatus that express nif genes constitutively with respect to ammonia were studied in order to define better the circuit that regulates nif gene transcription. One mutant class could be complemented in trans by a cosmid clone containing a wild-type gene (nifR5) defined by Tn5 inserts as being no longer than 1.6 kb. The nifR5 gene is unlinked to previously described nif genes. A second mutant class could not be complemented by the wild-type cosmid library. For one mutant in this class, a nifH::lac fusion was used to select further mutants that were Lac-. Only two of these could be complemented in trans to Lac+; the complementing gene was nifR4, which is analogous to the ntrA gene of enterobacteria. Both complemented strains were Nifc. Therefore these mutations do not bypass the need for the nifR4 gene product. A third class of constitutive mutant was found by selecting Nif+ revertants of a Nif- strain deleted for the nifR1 and nifR2 genes. The nifR1 and nifR2 genes are homologues of enterobacterial ntrC and ntrB genes, respectively. Not all of the Nif+ revertants were constitutive; some were regulated normally by ammonia. We suspect that the latter revertants use alternate Ntr systems to activate nif gene transcription, a suggestion consistent with the observation that numerous bands in Southern blots of total DNA of R. capsulatus are identified by Escherichia coli ntr gene probes.