L-Selectin (CD62L) mediates T-cell entry into lymph nodes. Whether the microenvironment modulates L-selectin expression of T cells during diapedesis and transit is unknown. Therefore, L-selectin expression was determined quantitatively on circulating T cells in blood, lymph nodes and thoracic duct by confocal laser scanning microscopy. We show that in contrast to leukocyte function-associated antigen-1 (CD11a/CD18) and ICAM-1 (CD54), L-selectin expression is cyclically expressed on recirculating T cells. It is reduced to approximately 30% of the blood value during entry across high endothelial venules. Within lymph nodes, CD4(+) T-cell subsets maintain reduced L-selectin expression at a similar level in all compartments (T-cell zone, B-cell zone and medulla). After exit, L-selectin is re-expressed to levels comparable to those of T cells in blood. Apparently, L-selectin levels are not only down-regulated during T-cell activation but also routinely reduced while transmigrating within lymph nodes. L-Selectin down-regulation seems to be ligand independent since it also occurs in the white pulp compartments of the spleen which lack classic L-selectin ligands such as GlyCAM-1 and CD34. In addition, T cells in non-lymphoid organs do not reveal reduced L-selectin levels. Thus, the ability of secondary lymphoid organs to reduce L-selectin expression of T cells prior to activation might be a prerequisite for their characteristic property to induce primary immune responses.