Escherichia coli DNA topoisomerase I requires Mg(II) as a cofactor for the relaxation of negatively supercoiled DNA. Mg(II) binding to the enzyme was shown by fluorescence spectroscopy to affect the tertiary structure of the enzyme. Addition of 2 mM MgCl2 resulted in a 30% decrease in the maximum emission of tryptophan fluorescence of the enzyme. These Mg(II)-induced changes in fluorescence properties were reversible by the addition of EDTA and not obtained with other divalent cations. After incubation with Mg(II) and dialysis, inductively coupled plasma (ICP) analysis showed that each enzyme molecule could form a complex with 1-2 Mg(II) bound to each enzyme molecule. Such Mg(II).enzyme complexes were found to be active in the relaxation of negatively supercoiled DNA in the absence of additional Mg(II). Results from ICP analysis after equilibrium dialysis and relaxation assays with limiting Mg(II) concentrations indicated that both Mg(II) binding sites had to be occupied for the enzyme to catalyze relaxation of negatively supercoiled DNA.