Cleavage of genomic DNA from Bradyrhizobium japonicum strain 3I1b110 by the restriction enzymes PmeI, PacI, and SwaI has been used together with pulsed-field gel electrophoresis and Southern hybridization to locate the nirK, norCBQD, and nosRZDFYLX denitrification genes on the chromosomal map of B. japonicum strain 110spc4. Mutant strains GRK13, GRC131, and GRZ25 were obtained by insertion of plasmid pUC4-KIXX-aphII-PSP, which carries recognition sites for the enzymes PacI, PmeI and SwaI, into the B. japonicum 3I1b110 nirK, norC and nosZ genes, respectively. Restriction of strain 3I1b110 genomic DNA with PacI, PmeI and SwaI yielded three, five and nine fragments, respectively. Pulsed-field gel electrophoresis of restricted mutant DNAs resulted in an altered fragment pattern that allowed determination of the position of the selected genes. Complementary mapping data were obtained by hybridization using digoxigenin-labeled B. japonicum 3I1b110 nirK, norBQD and nosZD as gene probes. The nirK, norCBQD and nosRZDFYLX genes were located close to the groEL(2), cycH and cycVWX genes, respectively, on the strain 110spc4 genetic map. In contrast to other denitrifiers, B. japonicum 3I1b110 denitrification genes were dispersed over the entire chromosome.