L-selectin mediates tethering and rolling of lymphocytes in high endothelial venules (HEV) of lymph nodes (LN) and of leukocytes at inflammatory sites. We used transgenic mice expressing varying levels of wild-type or a non-cleavable mutant form of L-selectin on T cells to determine the relationship between L-selectin density, tethering and rolling, and migration into LN. T cells expressing supraphysiological levels of either wild-type or non-cleavable L-selectin showed rolling parameters similar to C57BL/6 T cells in hydrodynamic flow assays and during rolling in Peyer's patch HEV. In contrast, PMA- or antigen-activated T cells and L-selectin(+/-) T cells expressing subphysiological levels of L-selectin showed reduced numbers of rolling cells with increased rolling velocity. Short-term homing studies showed that elevated expression of L-selectin above physiological levels had no effect on T cell migration to LN; however, low L-selectin expression resulted in reduced T cell homing to LN. Thus, T lymphocyte migration into LN is regulated by the density of cell surface L-selectin. In addition, there is a saturable density of L-selectin required for optimal homing to PLN in C57BL/6 mice, the L-selectin level on circulating naive T cells promotes optimal homing, and increased expression above saturating levels promotes no further increase in T cell recruitment.