The migratory properties of Lyt-2- and Lyt-2+ T cells in the mouse have been investigated. In short-term in vivo homing studies, Lyt-2- T cells localized consistently more efficiently than Lyt-2+ T cells in Peyer's patches (about 1.5 times as well), whereas both populations localized roughly equivalently in peripheral lymph nodes. These homing characteristics of Lyt-2- and Lyt-2+ subsets are largely independent of their organ source. The specificity of migration appears to be determined by selective recognition of organ-specific determinants on the endothelial cells of high endothelial venules (HEV), specialized venules that mediate the exit of migrating lymphocytes from the blood: In an in vitro assay of lymphocyte binding to HEV in lymphoid organ frozen sections, Lyt-2- cells constituted a significantly and consistently greater proportion of T cells binding to Peyer's patch HEV than of those binding to peripheral node HEV. The homing and HEV recognition preferences of the Lyt subsets are reflected in differences in their in situ representation in mucosal vs nonmucosal lymphoid organs, which suggests that the selective migration of these populations may be an important factor in determining the character of local immune responses.