The advent of whole genome sequences has brought with it a vast number of new potential proteins whose function is unknown. We describe an approach to sorting proteins into functional groups by comparative two-dimensional (2-D) gel mapping of cells grown under different physiological conditions. Computerized image analysis selects the proteins whose expression levels change significantly for subsequent identification by mass spectrometry. The protein groupings are further subdivided by directed alteration of their expression levels (e.g., by gene inactivation) and following the changes in the expression pattern of the mutants. We have applied this approach to study the regulation of micro- and anaerobically induced genes including the genes involved in nitrogen fixation in the symbiotic bacterium Bradyrhizobium japonicum. The results obtained show that in addition to the two known regulons controlled by the transcription factors NifA and FixK2, a third set of proteins may exist in B. japonicum which are induced by anaerobic conditions and are regulated independently. The approach can be applied generally and can be used to build up functional relationship maps of genomes. Protein identification by mass spectrometry was shown to be vital to the interpretation of the expression analysis since 15% of the 2-D gel spots contained more than one protein.