The identification of conventional dendritic cells (cDCs) in the intestinal mucosa has been hampered by the difficulties associated with isolating cells from the intestine and by the fact that overlapping markers have made it complicated to discriminate them accurately from other intestinal mononuclear phagocytes such as macrophages (MFs). Here we detail the protocols we have developed to isolate live leukocytes from both murine and human small and large intestines and describe reliable strategies which can be used to identify bona fide cDCs in such preparations.