Mesenteric lymph nodes (MLN) drain the gut where nutritive antigens and pathogens are encountered by lymphocytes of the gut-associated lymphoid tissue. We sought to determine how lymphocytes enter the MLN by studying mice double deficient for beta7 integrins and L-selectin. beta7/L-selectin double-deficient lymphocytes did not migrate into MLN. Most importantly, MLN formation was drastically impaired in beta7/L-selectin double-deficient mice. Lymphocyte numbers in MLN from beta7/L-selectin double-deficient mice were tenfold reduced compared to control mice. A high percentage of the few lymphocytes still detected in MLN from beta7/L-selectin double-deficient mice were CD44hi CD18hi, suggesting alternate migration pathways independent of L-selectin and beta7 integrin for these cells. We conclude that the combination of both molecules, L-selectin and beta7 integrin, is indispensable for MLN formation and that these molecules may mediate lymphocyte migration to MLN in a sequential and synergistical manner.