The activities of two forms (7.5 and 5.5 S) of DNA ligase and of terminal deoxynucleotidyl transferase (TdT) have been studied in human thymocytes at different ages from 20 weeks pre-natal to 37 years after birth. Thymocytes have been selected on the basis of relative size and antigenicity (CD3, OKT3 immunofluorescence) with the cell sorter. For DNA ligases, three kinds of cells can be distinguished: (i) large antigenically negative cells of 20-week fetus, expressing only the 7.5 S enzyme; (ii) large antigenically positive cells without ligase activity; (iii) smaller antigenically positive cells, expressing only the 5.5 S enzyme. This last form of enzyme is found after birth. With respect to TdT expressed in OKT3- 5 micron cells and to OKT3+ thymocytes, it is observed that 5.5 S DNA ligase is found in a thymocyte population distinct from cells expressing TdT. Therefore, these results allow us to consider the 5.5 S DNA ligase activity as an additional functional marker for thymocyte maturation in humans.