BACKGROUND AND AIMS: Mechanisms regulating M-cell formation are still poorly understood. In vitro studies showed that lymphocytes trigger the conversion of enterocyte cell lines into M cell-like cells on coculture, whereas in vivo their role in M cell differentiation is still elusive. Our aim was first to examine Rag-1-/- mice, lacking B and T lymphocytes, for the presence of intestinal M cells. Second, we investigated the role of lymphotoxin alphabeta signaling on M-cell formation, given its pivotal role in the development of mouse Peyer's patches. METHODS: Small intestines of Rag-1-/- mice, injected or not with soluble lymphotoxin beta receptor-immunoglobulin fusion protein, were analyzed morphologically using whole mount cytochemical staining, immunohistochemistry, and electron microscopy. RESULTS: Small Peyer's patch-like aggregates were found in Rag-1-/- mice in normal number and location. The overlying epithelium of such aggregates was reduced in size but still harbored M cells. In vivo neutralization of lymphotoxin beta-receptor signaling partially reduced the percentage of M cells. CONCLUSIONS: The absence of mature lymphocytes does not prevent the formation of M cells, indicating that the signaling molecules that support M-cell differentiation, such as lymphotoxin alphabeta, may also be supplied by non-B and non-T cells. Mature B lymphocytes, however, are required for the formation of a full-sized follicle-associated epithelium.