Lymphocyte migration into lymphoid organs is regulated by adhesion molecules including L-selectin and the β 7 integrins. L-selectin and α 4 β 7 are predominantly hypothesized to direct the selective migration of lymphocytes to peripheral lymph nodes and the gut-associated lymphoid tissues, respectively. To further characterize interactions between L-selectin and β 7 integrins during lymphocyte recirculation, mice deficient in both receptors (L-selectin/β 7 integrin−/−) were generated. The simultaneous loss of L-selectin and β 7 integrin expression prevented the majority of lymphocytes (> 95% inhibition) from attaching to high endothelial venules (HEV) of Peyer’s patches and other lymphoid tissues during in vitro binding assays. Moreover, the inability to bind HEV eliminated the vast majority of L-selectin/β 7 integrin−/− lymphocyte migration into Peyer’s patches during short-term and long-term in vivo migration assays (> 99% inhibition, p< 0.01). The lack of lymphocyte migration into Peyer’s patches correlated directly with the dramatically reduced size and cellularity (99% reduced) of this tissue in L-selectin/β 7 integrin−/− mice. High numbers of injected L-selectin/β 7 integrin−/− lymphocytes remaining in the blood of wild-type mice correlated with markedly increased numbers of circulating lymphocytes in L-selectin/β 7 integrin−/− mice. Loss of either L-selectin or the β 7 integrins alone resulted in significant but incomplete inhibition of Peyer’s patch migration. Collectively, the phenotype of L-selectin/β 7 integrin−/− mice demonstrates that these two receptors primarily interact along the same adhesion pathway that is required for the vast majority of lymphocyte migration into Peyer’s patches.