The basal presence of immunologically potent cells within the intestinal muscularis externa and their functional significance is unclear. Our aim was to investigate the basal distribution of various leukocyte populations within the rat jejunal muscularis. In addition, we sought to immunohistochemically phenotype the muscularis macrophage in jejunal whole-mounts, isolate these cells in primary culture, and investigate their ontogenesis. Macrophages form a regularly distributed network that expresses major histocompatibility complex class II, CD14 receptors, and a low level of CD11/CD18. The macrophages are activated by dissection and are present in fetal animals. Enriched macrophage cultures show a normal resident phenotype and remain present for weeks in dissociated muscularis cultures. The results also demonstrate the presence of neutrophils, monocytes, mast cells, and lymphocytes within the muscularis and suggest that the dense network of muscularis macrophages may be a potent resident trigger for inflammation in response to tissue injury or bacterial translocation.