Aim In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free-living and symbiotic conditions have been carried out. Methods and Results Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD-dependent oxidase activity when cells were incubated under micro-oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane-bound c-type cytochromes of 27 and 32kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E.meliloti cbb3 oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N-free nutrient solution during 3weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation. Conclusions The copy 1 of the fixNOQP operon is involved in E.meliloti respiration and growth under micro-oxic conditions as well as in the expression of the FixO and FixP components of the cbb3 oxidase present in free-living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis. Significance and Impact of the Study It is the first time that a functional analysis of the E.meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb3 oxidase operating in free-living micro-oxic cultures and in bacteroids of E.meliloti have been identified.