Human lymphocytes derived from various central and peripheral sources were separated on linear density gradients (LDG). Cells from individual density fractions were tested in parallel for: the capacity to form nonimmune rosettes with neuraminidase-treated SRBC, the number of surface-associated HTLA, and in vitro proliferative responses to mitogenic lectins and alloantigens. Heterogeneous density distribution profiles were obtained for all sources of human T cells and revealed an organ specificity. The various T cell density classes obtained from identical organs as well as the identical density classes of different sources revealed to some extent differences in their surface marker patterns and/or their in vitro reactivities. On the basis of the combined techniques at least two major subsets among thymocytes were identified that differed in both surface properties and functional capacities. Density classes of T cells from all peripheral sources were distinguished from thymocytes by a homogeneous lowered HTLA expression. Whereas clear-cut differences in the in vitro functional capacity were observed between the two thymocyte subsets, less striking but still significant differences were found to exist among the various density classes of peripheral T cells.