We have determined the nucleotide sequence of a segment of Co1E3 DNA coding for the carboxyl-terminal, catalytic peptide of colicin E3 and for the immunity protein. The end of the colicin E3 gene is separated from the beginning of the immunity gene by a nine-basepair intercistronic region, suggesting the two genes are expressed as a single transcriptional unit. The immunity gene is expressed, however, in E. coli strains containing Co1E3-pBR322 hybrid plasmids deleted for the 5'-end of the colicin gene. The DNA sequence also contains an unexpected open reading frame (ORF). This ORF is contained within the colicin gene and is in the +1 reading frame with respect to that gene. Plasmids containing the ORF, directed the synthesis of an 11 kilodalton protein in a cell-free, transcription-translation system.