Abstract Transducing derivatives of bacteriophage λ have been used to isolate small DNA restriction fragments which contain the primary bacterial att site (BOB′) for phage insertion and the secondary bacterial att site (ΔOΔ′) at galT. Four hybrid att sites, BOP′, POB, ΔOP′, and POΔ′, have also been isolated in small restriction fragments. Acrylamide-agarose or agarose gel electrophoresis profiles of two analogous isogenic sets of transducing phage were compared with profiles of wild-type λ to identify the att-containing fragments. Each set consisted of phages transducing bacterial DNA originating to the right (e.g., POB′ or POΔ′) or left (e.g., BOP′ or ΔOP′) of the prophage and a recombinant phage, transducing all of the bacterial DNA of both the rightward and leftward tranaducing lines (e.g., a regenerated BOB′ or ΔOΔ′). The att-containing fragment of each phage was identified by virtue of the fact that it was the only fragment which was unique to the gel profile of that phage. For the mutants transducing the primary bacterial att site, the restriction enzymes HindII + III and MboII were used sequentially to obtain fragments containing BOB′, POB′, and BOP′ of approximately 560, 580, and 340 base pairs in length, respectively. For the mutants transducing the secondary att site at galT, restriction enzymes HindIII, BamHI, EcoRI, and Hinfl were used to identify fragments containing ΔOΔ′, POΔ′, and ΔOP′ of approximately 755, 475, and 580 base pairs in length, respectively. These fragments can be isolated in sufficient quantity for biochemical studies on att site interactions and in sufficient purity for sequence analysis.