Nine isolates of Klebsiella pneumoniae belonging to capsular serotypes K1 and K2 were assayed for virulence in mice. Virulent isolates (50% lethal dose of less than 10(3) microorganisms) and avirulent isolates (50% lethal dose of over 10(6) microorganisms) were selected. Supplementation of a defined minimal medium with transferrin markedly reduced the growth of avirulent strains but had no significant effect on the growth of virulent strains. All isolates produced enterochelin, but only production of aerobactin could be correlated with virulence. The genes encoding aerobactin and its receptor protein were located on a 180-kilobase plasmid. They were cloned into the mobilizable vector pSUP202. Homology was demonstrated with the aerobactin operon of the Escherichia coli plasmid pColV-K30. Transfer of the recombinant plasmid pKP4 into an avirulent recipient enhanced virulence by 100-fold. These experiments demonstrated that aerobactin is an essential factor of pathogenicity in K. pneumoniae.