T lymphocytes utilize adhesion receptors in a regulated manner to interact with other cells and with components of the extracellular matrix. These cell-cell and cell-matrix interactions serve a critical role in T cell recognition of foreign Ag and in the migration of T cells to various anatomic sites in vivo. Thrombospondin is an extracellular matrix protein that is transiently expressed at high concentration in damaged and inflamed tissue. Given recent evidence implicating a role for the extracellular matrix in modulating T-cell migration and function, we analyzed T-lymphocyte interactions with thrombospondin. We show here that CD4+ T cells specifically adhere to thrombospondin predominantly via the 70 kDa core region of the thrombospondin molecule. Antibody blocking and affinity chromatography analysis suggest that T-cell adhesion to thrombospondin involves three distinct receptors: an activation-independent receptor that mediates adhesion of resting T cells, and the alpha 4 beta 1 and alpha 5 beta 1 integrins, which mediate a rapid increase in adhesion to thrombospondin upon activation. These three molecules appear to be novel thrombospondin receptors, as other receptors previously implicated in the adhesion of nonlymphoid cells to thrombospondin appear not to be involved in T-cell/thrombospondin interactions. The up-regulation of alpha 4 beta 1 and alpha 5 beta 1 functional activity upon activation is associated with the preferential adhesion of memory T cells to thrombospondin. Our results thus define three novel thrombospondin receptors, and provide additional evidence that extracellular matrix proteins play an important role in lymphocyte migration into, and retention in, inflammatory sites.