The tumor necrosis factor (TNF) family cytokines lymphotoxin (LT) alpha and LTbeta form heterotrimers that are expressed on the surface of activated lymphocytes and natural killer cells; LTalpha homotrimers can be secreted as well. Mice with a disrupted LTalpha gene lack lymph nodes (LN), Peyer's patches (PP), and follicular dendritic cell (FDC) networks and reveal profound defects of the splenic architecture. However, it is unclear which of these abnormalities is the result of the absence in LTalpha homotrimers or LTalphabeta heterotrimers. To distinguish between these two possibilities, a mouse strain deficient in LTbeta was created employing Cre/loxP-mediated gene targeting. Mice deficient in LTbeta reveal severe defects in organogenesis of the lymphoid system similar to those of LTalpha-/- mice, except that mesenteric and cervical LN are present in most LTbeta-deficient mice. Both LTbeta- and LTalpha-deficient mice show significant lymphocytosis in the circulation and peritoneal cavity and lymphocytic infiltrations in lungs and liver. After immunization, PNA-positive B cell clusters were detected in the splenic white pulp of LTbeta-deficient mice, but FDC networks were severely underdeveloped. Collectively, these results indicate that LTalpha can signal independently from LTbeta in the formation of PNA-positive foci in the spleen, and especially in the development of mesenteric and cervical LN.