Bacillus anthracis, the causal agent of anthrax, is a Gram-positive, spore-forming, aerobic, rod-shaped bacterium. Infection may result from intradermal inoculation, ingestion or inhalation of spores (Klein et al., 1966; Laforce et al., 1969; Friedlander et al., 1993). Humans may become infected by inhaling spore-contaminated aerosols generated by operations with domestic livestock. The transformation of a dormant spore into a vegetative cell, a key step in the pathogenic cycle of a spore-forming bacterium, enables bacteria to proliferate actively and to synthesize their virulence factors. B. anthracis is an extracellular pathogen. The vegetative forms are encapsulated and toxinogenic. The principal known B. anthracis virulence factors are an anti-phagocytic poly-γ-D-glutamic acid capsule (Green et al., 1985) and two exotoxins, the oedema (PA-EF) and lethal (PA-LF) toxins (Beall et al., 1962; Friedlander, 1986). The target cell binding domain, protective antigen PA (83 kDa), is common to two effector moieties: the oedema factor, EF (89 kDa), and the lethal factor, LF (90 kDa). Oedema factor is a calmodulin-dependent adenylate cyclase (Leppla, 1982) that induces an increase in intracellular concentrations of cyclic AMP in eukaryotic cells (Mock and Ullmann, 1993). It has been suggested that lethal factor is a Zn2+-metalloprotease (Klimpel et al., 1994). Anthrax lethal toxin causes death in laboratory animals (Ezzell et al., 1984) and has been implicated in anthrax pathogenesis (Stanley and Smith, 1961; Beall et al., 1962; Pezard et al., 1991). In vitro, the toxin causes cytolysis of primary macrophages (Mφs) and murine macrophage-like cells, such as J774A1 and RAW264.7 cells (Friedlander, 1986). At subcytolytic concentrations, lethal toxin induces the release of tumour necrosis factor (TNF-α) and interleukin-1β (IL-1β) from RAW264.7 cells (Hanna et al., 1993) and may generate reactive oxygen intermediates (ROIs) (Hanna et al., 1994). The anthrax lethal toxin has also been shown to have T-cell mitogenic activity (Guidi-Rontani et al., 1997). Therefore, the macrophage has a key role in B. anthracis pathogenesis: (i) as the first host cell interacting with B. anthracis spores via phagocytosis; (ii) as a cell that can induce host defence responses against the invading bacteria; and (iii) as the cell mediating cytotoxicity during anthrax infection. The pulmonary form of anthrax causes the rapid progression of the disease and often leads to death (Dalldorf and Beall, 1967; Fritz et al., 1995). Previous studies have suggested that inhaled spores are subjected to phagocytosis by macrophages and are carried by the lymphatic system to local mediastinal lymph nodes (Ross, 1957). The severity of infections with the pulmonary form of anthrax impelled us to investigate the involvement of the alveolar macrophages during the first stage of infection. Confocal scanning laser microscopy and image cytometry combined with a high-sensitivity, fluorescence-based reporter system made it possible to investigate host macrophage–pathogen interactions in single cells. In this study, we have shown that the alveolar macrophage is the primary site of B. anthracis germination in a murine inhalation infection model. We have also identified the cell compartment in which germination occurs and demonstrated the early onset of toxin gene expression.