We have previously demonstrated that the nifA promoter (nifAp) of Rhizobium meliloti is inducible under microaerobic conditions in the absence of alfalfa. Here we show that microaerobic activation of nifAp involves both cis- and trans-acting regulatory controls identical to those used symbiotically. The start site for nifA mRNA synthesis was found to be the same during symbiosis and microaerobiosis, and a deletion analysis of nifAp demonstrated that DNA between positions -62 and -45 is essential for induction. Mutants isolated as being unable to induce nifA microaerobically also were found to be defective in symbiotic nitrogen fixation with alfalfa. Such mutants form nodules that are equivalent cytologically to those induced by nifA::Tn5 mutants. Genetic and structural studies have localized the mutations to a cluster of fix genes 200 kilobases distant from the nod-nif region on the pSym megaplasmid [Renalier, M.-H., Batut, J., Ghai, J., Terzaghi, B., Gherardi, M., David, M., Garnerone, A.-M., Vasse, J., Truchet, G., Huguet, T. & Boistard, P. (1987) J. Bacteriol. 169, 2231-2238].