Abstract The immune response in many infections and to allografts is dependent on CD8+ cytotoxic T lymphocytes (CTL). Influx of CD8+ CTL from the blood has been documented during antigen challenge. We have previously found that a subset of CD8+ T cells from normal blood can migrate through endothelial cell monolayers in vitro. To further characterize migration-prone CD8+ T cells from normal blood, we examined the expression of CD28 and a restricted epitope of CD18/CD11a (S6F1), a CTL marker. Although normal blood CD8bright+ T cells were heterogeneous in their expression of CD28, three populations could be identified (CD28low, CD28moderate, and CD28high). CD8+ cells migrating across endothelial cell monolayers were enriched for CD8bright+ CD28high cells and a subset of CD8dim+ cells, which were CD28high. Both adherent and migrating CD8+ cells were exclusively (>95%) S6F1high. There was also preferential adhesion and migration of CD8+ cells expressing the low-molecular-weight form of the leukocyte common antigen, CD45RO. Cytokine activation of the endothelium did not significantly alter preferential migration of these subsets. These data suggest that certain subsets of CD8+ memory T cells in normal human blood are prone to, adhere to, and migrate through allogeneic endothelial cells and would thus be likely to be recruited to sites of antigen challenge.