As a model for T cell immigration into non-lymphoid tissue we set up an in vitro assay that would allow us to investigate the phenotype of T lymphocytes from peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) or peripheral blood (PBL) of mice, which were able to spontaneously migrate across unstimulated microvascular endothelium. The transendothelial migrating T cell population was enriched for T lymphocytes expressing a "recently activated/memory' phenotype: LFA-1/CD44/ICAM-1high, but also contained CD45RBhigh and LFA-1low T cells, which in the case of MLN T cells were phenotyped as CD4+ and thus characterized as naive T cells. Transmigrated T cells could be further distinguished from their original populations and from each other by their distinct but heterogeneous expression patterns for L-selectin, alpha 4 beta 7-integrin and PECAM-1. This observation suggests the presence of phenotypically different migratory T cells among MLN, PLN and PBL. Additional studies provided evidence that the capacity to migrate across unstimulated microvascular endothelium was a characteristic of a T cell population that could phenotypically be differentiated from activated T cells. The endothelial cells were found to play an active role in selecting the traversing T cell population, as they controlled the number and phenotype of spontaneously transmigrating T cells. Our studies suggest that the capacity to transmigrate across unstimulated microvascular endothelium and hence to immigrate into non-lymphoid tissue is owned by a phenotypically heterogeneous T cell population, which is enriched for memory T cells but not devoid of naive T cells.