e Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received tremendous attention due to their health-promoting properties. We report the development of two dual-color luciferase-producing Lactobacillus (Lb.) plantarum and Lactococcus (Lc.) lactis strains for noninvasive simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional expression of the red luciferase mutant (CBRluc) from Pyrophorus plagiophthalamus in Lb. plantarum NCIMB8826 and Lc. lactis MG1363 (C. Daniel, S. Poiret, V. Dennin, D. Boutillier, and B. Pot, Appl Environ Microbiol 79:1086 -1094, 2013, http://dx.doi.org/10.1128/AEM.03221-12). In this study, we determined that CBRluc is a better-performing luciferase for in vivo localization of both lactic acid bacteria after oral administration than the green click beetle luciferase mutant construct developed in this study. We further established the possibility to simultaneously detect red-and green-emitting lactic acid bacteria by dual-wavelength bioluminescence imaging in combination with spectral unmixing. The difference in spectra of light emission by the red and green click beetle luciferase mutants and dual bioluminescence detection allowed in vitro and in vivo quantification of the red and green emitted signals; thus, it allowed us to monitor the dynamics and fate of the two bacterial populations simultaneously. Persistence and viability of both strains simultaneously administered to mice in different ratios was studied in vivo in anesthetized mice and ex vivo in mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to simultaneously study the interactions and potential competitions of different targeted bacteria and their hosts. L actococci and lactobacilli are lactic acid bacteria (LAB) that have been used for thousands of years for the production and preservation of fermented food, such as milk, vegetables, and meat. Some specific strains are commercialized as probiotics and claimed to have health-promoting properties (1). The gastrointestinal tract (GIT) is the most important field of activity of LAB, although distal effects outside the gut have been described (2). Thus, it is important to understand the interactions of the administered bacteria with their host GIT system. LAB are able to survive and adapt to the GIT conditions, as shown by comparative and functional genomic characterization of various human isolates, essentially lactobacilli (for a review, see references 2 and 3). Some LAB, when present in the GIT of mice or humans, express a number of common characteristics that may relate to their intestinal niche adaptation (3, 4). However, direct in vivo tracking of these actions in terms of both spatial and temporal evolution would allow a better understanding of the survival and metabolic activities of these LAB in the gut.In the past decade, bioluminescence imaging (BLI) has become essential for in vivo noninvasive monitoring of biological processes (4). The technique relies on ...