A new colicin, as defined by cross-immunity tests against the colicin reference set, has been identified from a uropathogenic Escherichia coli isolate (strain 2424), collected from a pyelonephritis patient at Christchurch Hospital. This toxin, colicin 24, is active against E. coli and Salmonella typhimurium, and the genes involved in colicin 24 production, transport, and immunity were found to reside on a 43.54-kb conjugative plasmid, p24-2. Mutagenesis using mini-Tn10 has identified two consecutive EcoRI fragments on p24-2 that contain all the colicin 24 determinants. A 25.14-kb BamHI-HindIII fragment containing this region was cloned from a mutant with a normal colicin phenotype into pBR322 to form the recombinant plasmid pGOB34. Subcloning of pGOB34 has resulted in an 8.7-kb fragment containing all the colicin 24 determinants being cloned into pUC18 to form pGOB342.