Integrase (Int) carries out the cutting and resealing of attachment (att) site DNA via a covalent Int-DNA intermediate. A family of synthetic substrate DNAs was designed to accumulate Int-DNA intermediate. Int-DNA intermediates accumulated by half substrate was analyzed by SDS- KCI precipitation and restriction digestion. The results showed that Int-half DNA intermediate was circular and contained covalently bound Int molecule. Int-DNA intermediates were also trapped with three other kinds of synthetic substrates.