The lack of probes defining leukocyte subpopulations has restricted ontogenetic studies of the opossum gut. We report for the first time the organization of the gut cellular immune components using species cross-reactive antibodies. Mouse monoclonal antibodies against human HLA-DR were used together with immunocytochemistry to demonstrate MHC class II-like antigens in the opossum Peyer's patches (PP). Positive staining was obtained in the M cell and enterocytes comprising the follicular-associated epithelium (FAE). Rabbit polyclonal antibody against human CD3 stained opossum thymocytes and T-cell dependent areas of spleen, lymph node, and PP interfollicular zones, but failed to stain intraepithelial lymphocytes in the FAE. In contrast rabbit polyclonal antibody against human IgA stained B-cell immunocytes and plasma cells present in the M-cell lateral invaginations. It is surmised that B-cell activation could occur in the opossum M-cell niches by thymus independent antigens, bypassing T-helper-cell function.