Lymphocyte migration into lymphoid organs is regulated by tissue-specific adhesion molecules such as L-selectin and the alpha4beta7 integrin. Whether L-selectin also regulates lymphocyte subset-specific migration into specific lymphoid tissues was examined in this study by comparing the migration of CD4+ T cells, CD8+ T cells, and B cells from L-selectin-deficient and wild-type mice. T cells were the predominant lymphocyte subset entering PLN, MLN, Peyer's patches, and spleen during short term (1-h) migration assays. However, both B cell and CD4+ and CD8+ T cell entries into PLN, MLN, and Peyer's patches were dramatically impaired (73-98%) by loss of L-selectin. Lymphocyte expression of alpha4beta7 integrin did not compensate for the loss of L-selectin, since both B and T cells predominantly migrated into the spleen in the absence of L-selectin. The more efficient migration of T cells into peripheral lymphoid tissues relative to that of B cells was partly explained by the finding that T cells expressed L-selectin at 50 to 100% higher levels than B cells. In addition, a 50% reduction in L-selectin expression by lymphocytes from hemizygous L-selectin(+/-) mice resulted in a 50 to 70% decrease in short term lymphocyte migration into peripheral lymphoid tissues relative to that of wild-type lymphocytes. Thus, the differential migration of T and B lymphocyte subsets to lymphoid tissues is regulated in part by subset-specific differences in L-selectin expression levels.