Lymphotoxin (LT) provides a critical signal for the genesis of lymph nodes (LN) in mice. Here we show that mice treated in utero with LTb-R-Ig, which binds to the membrane LTa1b2 heterotrimer, lacked most LN, yet retained a set of mucosal surface draining LN. Since mice genetically deficient in LTa lack all LN, including the mucosal set, we hypothesize that a novel LTa-dependent pathway controls their genesis. This novel set of mucosal LN cannot be discriminated on the basis of addressin expression. The discovery of LN in mice treated with LTb-R-Ig fusion protein in utero allowed us to compare the roles of membrane LTab or soluble LTa/tumor necrosis factor (TNF) in the development of cellular organization in LN and spleen. Our results indicate that both membrane LTab and soluble LTa/TNF mediate T-B cell segregation and the organization of B cell follicles in spleen and LN. Interestingly, while antagonism of membrane LTab or soluble LTa/TNF prevented germinal center (GC) formation in spleen, antagonism of soluble LTa/TNF had no effect on LN formation. The data suggest that multiple LT/TNF ligands control B cell follicle organization in the spleen and LN of adult mice, and that the requirements for LT/TNF ligands in GC formation are distinct in the different lymphoid organs.