Isolated lympheid follicles (ILFs) are organized lymphoid structures in the small intestine. ILFs were recently identified in the murine small intestine; however, the function of ILFs is unknown. To better understand ILFs and the role they play in the intestinal immune response, we have examined the composition of ILFs, the factors that are involved in the genesis of ILFs, and the ability of ILFs to support antigen-specific immunoglobulin production. We found that ILFs contain predominantly B-2 B lymphocytes, and CD4+ TCRβ+ T lymphocytes. Similar to the formation of Peyer's patches (PPs), lymphotoxin β receptor (LTβR)-dependent events are required for ILF formation; however, the timing of these events and the cellular source of LT differ. ILF formation can occur de novo in response to luminal stimuli and requires LT-safficient B lymphocytes and TNF receptor I function for full maturation. The epithelium over ILFs resembles the PP follicle-associated epithelium, as M cells are present and pathogens such as Yersinia can be bound and taken up into the underlying follicle. Total fecal IgA production is not augmented in animals possessing ILFs; however, the production of antigen-specific IgA is increased in animals possessing ILFs orally challenged with Salmonella typhimurium. Similar to PPs, ILFs can support antigen-specific IgA production following oral immunization. These findings support the concept that ILFs are formed in response to mucosal challenges, and may play a physiological role in the production of antigen-specific intestinal IgA. © 2004 New York Academy of Sciences.