Rifampicin-encapsulated liposome suspensions were prepared by a chloroform-film method and converted to dry powders by freeze-drying with mannitol as a cryoprotectant. The liposome suspension had multilamellar nanovesicles with 50% rifampicin encapsulation. The liposome dry powder comprised particles with a mass median aerodynamic diameter of 3.4 mum, with 60% present as a fine particle fraction. Rifampicin-encapsulated liposomes were evidently nontoxic to respiratory associated cells, including bronchial epithelial cells, small airway epithelial and alveolar macrophages (AMs). Furthermore, the liposomes did not activate AMs to produce interleukin-1 beta, tumor necrosis factor-alpha, and nitric oxide at a level that would cascade to other inflammatory effects. The minimum inhibitory concentrations against Mycobacterium bovis was 0.2 and 0.8 microM for liposomes containing rifampicin and free rifampicin, respectively. The less negatively charged reconstituted liposome displayed the greatest activity against intracellular growth of M. bovis.