We constructed mutant strains of Francisella tularensis biotype novicida by insertional mutagenesis with a kanamycin resistance (KmR) cassette. One mutant, KEM7, was defective for survival in macrophages in comparison with the wild-type (WT) strain and a random insertion strain, KEM21. While all three strains exhibited intracellular growth, the number of viable KEM7 present after 24-48 h of infection was approximately 10 times less than that of WT or KEM21. This observation was apparently due to a reduced number of viable KEM7 associated with the macrophages one hour after phagocytosis. KEM7 was approximately 3 times more susceptible than WT or KEM21 to killing by the products of the xanthine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also found to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 1,000- to 10,000-times less than that of either KEM21 or WT. DNA sequence analysis at the KmR insertion site suggested that the F. tularensis homologue of minD had been interrupted. Western immunoblot analysis confirmed the presence of a MinD homologue in F. tularensis WT and KEM21, and demonstrated its absence in KEM7.