The origin of replication of the Escherichia coli chromosomal DNA binds with high affinity to outer membrane preparations. This specific binding requires a 463-base-pair region of origin DNA between positions -45 and +417 of the oriC map. We show that binding does not require the presence of adjacent regions. From further analysis, we conclude that more than one binding site resides within the 325-base-pair fragment between positions +38 (BamHI) and +417 (XhoI). When this fragment is cut, two pieces bind with high affinity and one binds with lesser affinity. The binding ability of one of the high affinity sites is abolished by cutting it at position +92 with BamHI.