Abstract A revised sequence in the region immediately upstream from the rop gene of pBR322 is reported. Two base pairs in the accepted sequence do not exist in the plasmid DNA. Specifically, a TA base pair is missing at sequence coordinate 1893 [Sutcliffe, Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 77–90] and an AT base pair is missing at position 1915, giving a total size for pBR322 of 4361 bp. These changes are in a potential translation initiation sequence and probably reflect errors in the original sequence rather than recent evolution of the plasmid.