L-selectin mediates rolling of lymphocytes in high endothelial venules (HEVs) of peripheral lymph nodes (PLNs). Cross-linking of L-selectin causes proteolytic shedding of its ectodomain, the physiological significance of which is unknown. To determine whether L-selectin shedding regulates lymphocyte migration, a mutant form that resists shedding (LdDeltaP-selectin) was engineered. Transgenic mice expressing either LDeltaP or wild-type (WT) L-selectin on T cells were crossed with L-selectin knockout (KO) mice. The cellularity and subset composition of secondary lymphoid organs did not differ between LDeltaP and WT mice, however, they were different from C57BL/6. Plasma levels of soluble L-selectin in LDeltaP mice were reduced to <5% of WT and C57BL/6 mice. The rolling properties of T lymphocytes from LDeltaP and WT mice on immobilized L-selectin ligands were similar. Furthermore, similar numbers of LDeltaP and WT T lymphocytes were recruited from the bloodstream into PLNs in mice, although LDeltaP T cells transmigrated HEVs more slowly. WT, but not LDeltaP-selectin, underwent rapid, metalloproteinase-dependent shedding after TCR engagement, and LDeltaP T cells retained the capacity to enter PLNs from the bloodstream. These results suggest that the ability to shed L-selectin is not required for T cell recirculation and homing to PLNs. However, L-selectin shedding from antigen-activated T cells prevents reentry into PLNs.