Abstract The bacteriophage P1 recombinase Cre mediates site-specific recombination between loxP sites. The loxP site consists of two 13 base-pair inverted repeats separated by an eight base-pair spacer region. When DNA containing the loxP site is incubated with Cre, specific cleavages occur within the spacer region, creating a six base-pair staggered cut. The cuts are centered on the axis of dyad symmetry of the loxP site, resulting in a 5′ protruding terminus: 5′ A↓T-G-T-A-T-G C 3′ T A-C-A-T-A-C↑G At the point of cleavage, Cre becomes covalently attached to a 3′ PO 4, and produces a free 5′ OH. A series of experiments were carried out in which a radioactively labeled loxP site is recombined with an unlabeled loxP site to locate the point at which strand exchange takes place during recombination. The points of strand exchange coincide with the sites at which Cre cleavage of the DNA backbone had been detected.