I have derived a complete restriction map of pBR322 from the total nucleotide sequence of the plasmid. Most of the restriction sites also have been demonstrated empirically. The exact sizes of all restriction fragments and the relative positions of the cuts are presented. These fragments can serve as accurate DNA size markers from small pieces up to the 4362 base pair length of pBR322. Inserts cloned in this vector may be characterized easily using this data.