Salmonella typhimurium, like many other Salmonella serovars, harbours a large plasmid required for mouse virulence and growth of bacteria in host cells. The nature of one virulence-abolishing Tn5 insertion (zzx-2556::Tn5) in the plasmid was characterized. A 3.7 kb insert harbouring this region was cloned in pBR325. Plasmid-directed protein synthesis in minicells indicated that the transposon had eliminated the expression of a 70 kDa protein encoded by the virulence plasmid. Nucleotide sequence analysis of the corresponding wild type DNA region showed a 1773 bp open reading frame (mkaA), encoding a protein of 591 amino acid residues and a predicted molecular mass of 60.6 kDa; zzx-2556::Tn5 was situated within mkaA.