Endonuclease I is a DNA junction-selective resolving enzyme from bacteriophage T7. Using a nuclease-defective mutant that retains normal binding to DNA we show that the protein binds to four-way DNA junctions as a dimer, in common with other junction-resolving enzymes studied. Gel filtration and chemical crosslinking indicate that endonuclease I also exists in free solution as a dimer together with a tetramer and higher molecular mass aggregates. However, in marked contrast with other junction-resolving enzymes, there is no detectable subunit exchange under normal conditions. Only by exposure to 6 M urea could we induce subunit exchange, and this was used to generate heterodimeric species containing one active and one inactive subunit. Using a supercoil-stabilised cruciform substrate we demonstrate that an active subunit of endonuclease I can act as a junction-specific nuclease in a heterodimeric combination with an inactive subunit. However, the two subunits of a fully active homodimeric enzyme each cleave the phosphodiester backbone of a cruciform within the lifetime of the DNA-protein complex.