Immunogold preparations of Escherichia coli, using anti-GyrA and anti-GyrB antibodies to the subunits of DNA gyrase, showed clear labelling with both secondary antibody and protein A-gold conjugates. Both proteins were located mainly in the cytoplasm, with typically less than 10% in the nucleoid. This partitioning of gyrase proteins between nucleoid and cytoplasm was nonrandom and was consistently observed for a range of different cell preparations. Total gold particle counts were highly variable but suggested levels of at least 1000-3000 molecules per cell for both GyrA and GyrB. Sequential treatment with both anti-GyrA and anti-GyrB monoclonal antibodies resulted in simultaneous labelling of both proteins and revealed no clear association between the two groups of molecules. Treatment of cells with chloramphenicol caused marked changes in nucleoid conformation, but no reduction in cytoplasmic labelling of gyrase proteins. On the assumption that gyrase complexes within the nucleoid are not differentially masked from the monoclonal antibodies, the results obtained in this study suggest that most of the gyrase proteins are not associated with either central nucleoid DNA or cytoplasmic loops of peripheral single-stranded DNA, but are distributed randomly throughout the cytoplasm.