The uptake and persistence of Bordetella bronchiseptica was characterized in murine phagocytes by using a novel bioluminescence-based reporter system. A mini-Tn5 promoter probe carrying the intact lux operon from the terrestrial bacterium Photorhabdus luminescens which allowed measurement of light output without the addition of exogenous substrate was constructed. It was used to create a pool of bioluminescent fusion strains of B. bronchiseptica. The internalization and persistence in murine macrophages of a constitutive bioluminescent strain of B. bronchiseptica was monitored by luminometry and by fluorescence and electron microscopy. The number of bacteria internalized, in a microfilament-dependent process, by a mouse macrophage-like cell line after 2 h was approximately 1% of the inoculum for several different multiplicities of infection (MOI). At an MOI of <500:1 (bacteria to macrophages), viable numbers of intracellular bacteria declined over a 4-day period. However, at an MOI of 500:1, long-term survival was enhanced, with viable bacteria recovered up to 4 days postinfection with little decline in numbers, indicating that a critical population size may have been essential for intracellular persistence. No evidence of macrophage killing by intracellular bacteria was detected over the 4-day period. Intracellular bioluminescent B. bronchiseptica organisms in mouse peritoneal cells were detected at 24 and 48 h after intraperitoneal injection of mice. Bioluminescence is shown to act as a convenient real-time technique for monitoring of intracellular survival of B. bronchiseptica in vitro and may provide a suitable means for examining the role of long-term intracellular survival of the bacterium in the host.