The purpose of this paper was to study the heterogeneity of human thymocytes and leukemic cells of the T-cell line MOLT-3 by velocity sedimentation. Analysis of the subpopulations of thymocytes demonstrated that they represent a heterogeneous population of cells with respect to their size, proliferative activity, and presence and quantities of terminal deoxynucleotidyl transferase and human thymus leukemia-associated antigen, a thymic isozyme of adenosine deaminase (HThy-L/ADA). Only a minor subpopulation of thymocytes (large cells) was in active cycle. The highest level of HThy-L/ADA was associated with the main subpopulation of thymocytes sedimenting at 3 to 4 mm/hr while low amounts of the HThy-L/ADA antigen (enzyme) were found in the minor fractions of the small and large cells. The distribution of terminal deoxynucleotidyl transferase-positive cells indicated that most, but not all, thymocytes contain the enzyme. Analysis of the T-cell line MOLT-3 showed that these cells could be separated into subpopulations with different biochemical and biological properties. More than one subpopulation of cells was capable of DNA synthesis. In contrast to the thymocytes, all fractions of MOLT-3 cells contained high amounts of HThy-L/ADA. The proportion of terminal deoxynucleotidyl transferase-positive cells as a function of sedimentation velocity was also quite constant although there was a slight but reproducible drop in the percentage of these cells in the slowly sedimenting fractions. The percentage of cells with receptors for sheep erythrocytes also remained high in fractions separated on the basis of size, although a consistently higher percentage was found in smaller cells. These studies indicated that thymus cells as well as the malignant T-cell line MOLT-3 can be separated on the basis of sedimentation velocity into subpopulations with different biological and biochemical properties. The data also indicated that the heterogeneity of MOLT-3 line cannot be explained solely on the basis of volume changes due to cell cycle, suggesting that they may represent heterogeneous populations of cells.