Thymus-derived cell populations were characterized by sedimentation velocity in the Earth's gravitational field, by adherence properties, and in terms of the thymus-specific antigen RTLA. T cells, responding to mitogens, could be shown to be a subpopulation of RTLA-bearing cells and to have a relatively large volume. The size distribution of mitogen-responsive cells from different individuals showed some variation, but in general large spleen cells took up less thymidine per 10(6) cells than did large cells from other lymphoid organs. Upon stimulation with concanavalin A (con A) small popliteal lymph node cells took up more thymidine per 10(6) cells than did small thymus cells. The corresponding small cells of mesenteric lymph node and spleen took up intermediate quantities of thymidine. Thymus-derived cells that were resistant to complement-mediated cell kill with RTLA antiserum showed greater responsiveness to con A than did the original cell preparations. Non-adherent cells incorporated more thymidine when stimulated with phytohaemagglutinin (PHA) than when they were stimulated with con A. Lightly adherent cells were relatively more responsive to con A stimulation and firmly adherent cells were less responsive to both. The findings were discussed in terms of the density of RTLA on the membranes of T cells. In adherence and in sedimentation velocity fractionation, the relative yield of nucleated cells always exceeded the yield of PHA- and con A-responsive cells. The differential separation of mitogen responsive and of co-operating cells were considered as a possible cause of this deficit.