Lactic acid bacteria, especially lactobacilli, are common inhabitants of the gastrointestinal tract of mammals, for which they have received considerable attention due to their putative health-promoting properties. In this study, we describe the development and application of luciferase-expressing Lactobacillus plantarum and Lactococcus lactis strains for noninvasive in vivo monitoring in the digestive tract of mice. We report for the first time the functional in vitro expression in Lactobacillus plantarum NCIMB8826 and in Lactococcus lactis MG1363 of the click beetle luciferase (CBluc), as well as Gaussia and bacterial luciferases, using a combination of vectors, promoters, and codon-optimized genes. We demonstrate that a CBluc construction is the bestperforming luciferase system for the noninvasive in vivo detection of lactic acid bacteria after oral administration. The persistence and viability of both strains was studied by bioluminescence imaging in anesthetized mice and in mouse feces. In vivo bioluminescence imaging confirmed that after a single or multiple oral administrations, L. lactis has shorter survival times in the mouse gastrointestinal tract than L. plantarum, and it also revealed the precise gut compartments where both strains persisted. The application of luciferase-labeled bacteria has significant potential to allow the in vivo and ex vivo study of the interactions of lactic acid bacteria with their mammalian host. L actococci and lactobacilli are lactic acid bacteria (LAB) which comprise a large variety of microorganisms that are applied to industrial and artisanal dairy, meat, or plant fermentation. Some selected strains are believed to be beneficial to human and animal health and are marketed as probiotics. Their major field of activity is believed to be the gastrointestinal tract (GIT), although many secondary effects outside the gut have been described. Therefore, it is important to understand the interactions between the bacteria administered and the host intestinal system. Unfortunately, the survival and metabolic activities of these bacteria in the GIT often remain uncertain, impairing our understanding of all the beneficial health effects of these organisms. LAB strains may adapt to the conditions in the intestinal tract, as shown by genetic screening and complementary studies that identified genes in Lactobacillus plantarum and Lactobacillus reuteri, that are specifically induced in the GIT of mice or by whole-genome transcriptome profiling of L. plantarum and Lactobacillus johnsonii in mice or humans (for a review, see reference 1). A better understanding of the survival and metabolic activities of LAB would be facilitated by direct in vivo monitoring of these processes in terms of both spatial and temporal evolution.Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo (2). In vivo bioluminescence imaging (BLI) represents one of the most outstanding uses of the technology b...