The structure of gamma delta resolvase complexed with a 34 bp substrate DNA has been determined at 3.0 A resolution. The DNA is sharply bent by 60 degrees toward the major groove and away from the resolvase catalytic domains at the recombination crossover point. The C-terminal one third of resolvase, which was disordered in the absence of DNA, forms an arm and a 3-helix DNA-binding domain on the opposite side of the DNA from the N-terminal domain. The arms wrap around the minor groove of the central 16 bp, and the DNA-binding domains interact with the major grooves near the outer boundaries of the binding site. The resolvase dimer is asymmetric, particularly in the arm region, implying a conformational adaptability that may be important for resolvase binding to different DNA sites in the synaptosome. It also raises the possibility of a sequential single-strand cleavage mechanism.