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Traditional techniques analyzing mouse colitis are invasive, laborious, or indirect. Development of in vivo imaging techniques for specific colitis processes would be useful for monitoring disease progression and/or treatment effectiveness. The aim was to evaluate the applicability of the chemiluminescent probe L-012, which detects reactive oxygen and nitrogen species, for in vivo colitis imaging.Two genetic colitis mouse models were used; K8 knockout (K8−/−) mice, which develop early colitis and the nonobese diabetic mice, which develop a transient subclinical colitis. Dextran sulphate sodium was used as a chemical colitis model. Mice were anesthetized, injected intraperitoneally with L-012, imaged, and quantified for chemiluminescent signal in the abdominal region using an IVIS camera system.K8−/− and nonobese diabetic mice showed increased L-012-mediated chemiluminescence from the abdominal region compared with control mice. L-012 signals correlated with the colitis phenotype assessed by histology and myeloperoxidase staining. Although L-012 chemiluminescence enabled detection of dextran sulphate sodium–induced colitis at an earlier time point compared with traditional methods, large mouse-to-mouse variations were noted. In situ and ex vivo L-012 imaging as well as [18F]FDG-PET imaging of K8−/− mice confirmed that the in vivo signals originated from the distal colon. L-012 in vivo imaging showed a wide variation in reactive oxygen and nitrogen species in young mice, irrespective of K8 genotype. In aging mice L-012 signals were consistently higher in K8−/− as compared to K8+/+ mice.In vivo imaging using L-012 is a useful, simple, and cost-effective tool to study the level and longitudinal progression of genetic and possibly chemical murine colitis.