Nucleotide sequencing of part of the plasmid pCHAP4, which encodes the ca. 42,000 Da putative poreforming colicin N, confirmed previous results indicating that the colicin N immunity gene (cni) and the colicin release or lysis gene (cnl) are located immediately downstream from the colicin N structural gene (cna) in the order cna-cni-cnl. The cni gene is transcribed in the opposite direction to cna and probably encodes an Mr 15239 Da protein. The putative immunity protein was detected among the [35S]methionine-labelled proteins produced by minicells carrying cni cloned under lac promoter control, and when the gene was subcloned into expression vectors under the control of a bacteriophage T7 promoter. Deletion of the region immediately upstream from cni completely abolished colicin N immunity, presumably because the natural promoter had been deleted. cnl is in the same operon as cna, and encodes a typical Col plasmid pro-lysis protein comprising a signal peptide and a 34 residue mature polypeptide with high homology to all but one of the other known Col lysis proteins, including the fatty acylated amino-terminal cysteine residue which was specifically labelled with 3H-palmitate. Cell fractionation studies indicated that the cnl gene product was located predominantly in the outer membrane.