Melioidosis is an infectious disease caused by the saprophytic gram-negative rod Burkholderia pseudomallei. The aim of this study was to establish and characterize a murine model of melioidosis to provide a basis for further investigations on the pathogenesis of the disease. After intravenous infection with B. pseudomallei, C57BL/6 mice were found to be significantly more resistant than BALB/c mice. There was a marked organotropism of B. pseudomallei for the spleen and liver in both strains of mice, with the highest bacterial load in the spleen. Electron microscopic investigations of the spleen clearly demonstrated intracellular replication within membrane-bound phagosomes. Electron micrographs of the liver provided evidence that B. pseudomallei-containing phagosomes in hepatocytes fuse with lysosomes, leading to degradation of bacteria. In both strains of mice, the course of infection was highly dependent on the infective dose and the bacterial strain used, ranging from death within a few days to death after several weeks. In comparison with BALB/c mice, the bacterial counts in C57BL/6 mice were decreased 12 h after infection, which is suggestive of an innate immune mechanism against B. pseudomallei in this early phase of infection contributing to the lower susceptibility of C57BL/6 mice. BALB/c mice developed a more pronounced lymphopenia, granulocytosis, and splenomegaly at a lower infective dose compared to C57BL/6 mice. Analysis of the antibody response against B. pseudomallei 11 days after infection revealed a significantly higher immunoglobulin G2A (IgG2a)/IgG1 ratio in C57BL/6 mice than in BALB/c mice, indicating that a T helper type 1 immune response is associated with resistance to infection with B. pseudomallei.