The analysis of seven differentiation markers following incubation with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined in the human leukemic T-cell line MOLT-3. Significant changes were observed in the activity of the markers terminal deoxynucleotidyl transferase (TdT). spontaneous proliferation and the ability of these cells to bind sheep erythrocytes. Levels of human thymus-leukemia-associated antigen (HThy-L) recently identified as a low molecular weight form of adenosine deaminase (ADA), were reduced by about 50%. No significant changes were observed in ecto-5'-nucleotidase [5'-NT) activities, in the proliferative response to PHA, or in the expression of IA-like antigens. These data and the time kinetics of the changes suggest that following incubation of these T-lymphoblasts with TPA there is a sequential loss of TdT, loss of the capacity for spontaneous proliferation, and the appearance of receptors for sheep erythrocytes. Subsequently there is a decrease in the level of HThy-L/ADA. This sequence appears to follow that proposed for prethymic precursor T-cell differentiation following activation with thymic epithelium.