L-selectin regulates lymphocyte migration by mediating lymphocyte attachment to high endothelial venules of peripheral lymph nodes (PLN). L-selectin-deficient mice display a 70 to 90% reduction in the number of lymphocytes within PLNs and a 30 to 55% increase in spleen cellularity. The altered distribution of lymphocyte subpopulations in L-selectin-deficient mice resulted in significantly elevated serum IgM and IgG1 levels and augmented humoral immune responses to T cell-independent and T cell-dependent Ags following i.p. immunization. By contrast, s.c. immunization of L-selectin-deficient mice with a T cell-dependent Ag resulted in serum IgM responses that were 40% lower when compared with wild-type littermates on day 7, while primary IgG responses were essentially absent. Most serum Ig responses were normal by day 14 and secondary responses were higher in L-selectin-deficient mice. Although lymphocytes from L-selectin-deficient mice were unable to enter Ag-stimulated lymph nodes through high endothelial venules, the cellularity of draining lymph nodes increased significantly following immunization. Germinal centers developed rapidly following immunization, although PLNs of L-selectin-deficient mice contained few follicles. Germinal centers within PLNs and the spleen were also consistently much larger and more well defined in L-selectin-deficient mice when compared with wild-type littermates. These results confirm that lymphocyte migration plays an important role in the initiation of humoral responses and demonstrate complementary and overlapping roles for the spleen and other peripheral lymphoid tissues in the generation of immune responses.