The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LTα-deficient mice lack all lymph nodes and Peyer’s patches, mice deficient in LTβ retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LTβ-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LTβ-deficient mice responsible for draining the genital mucosa. In the majority of LTβ-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LTβ-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LTβ-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LTβ-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.