The localization of monoclonal anti-Thy-1 binding in mouse thymus, spleen, and lymph nodes was studied at early intervals after intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) injection of a single dose of the monoclonal antibody (MAb). Five minutes after i.v. injection, anti-Thy-1 was bound to cortical thymocytes surrounding capillaries in the thymic cortex, to thymic cells beneath the thymic capsule and to medullary thymocytes around venules of the thymus medulla. When anti-Thy-1 was injected i.p. or s.c. the MAb was first deposited in capillary walls in the thymus cortex and did not appear on thymocytes outside of capillaries until 60 min after injection. These findings suggest that thymic cortical capillaries are permeable for anti-Thy-1 MAb contrary to the generally accepted principle of a blood thymus barrier to antigens in thymic cortex. Some cortex capillaries also became permeable for peroxidase when injected 15 min after anti-Thy-1 MAb. Anti-Thy-1 MAb penetration into spleen white pulp and lymph node paracortex occurs along the circulatory pathway of the vascular system in the spleen and of lymphatics in lymph nodes. But those lymphocytes with a strong anti-Thy-1 MAb loading always appeared along the pathways of lymphocyte circulation indicating that the most intense contact between anti-Thy-1 MAb and T-lymphocytes occurs not in the lymphatic organs but during the intravascular period of recirculation of lymphocytes.