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5-Ethynyl-2′-deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a “terminal” state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD. In vivo pulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.