The combination of centrifugal elutriation as an efficient and reproducible method to separate thymocytes by size, micromethods to assess purine interconversion enzymes, and assessment of purine (deoxy)nucleoside inhibition of mitogen responses enabled us to study purine metabolism at the intrathymic level. Out of six fractions, four (nos. 3-6), containing medium- and large-sized lymphocytes, showed a proliferative response after stimulation with phytohaemagglutinin (PHA). In fractions 1-6 the number of cells with an immature immunological phenotype gradually decreased, and cells with the phenotype of mature cells gradually increased. The enzyme activity ratio of adenosine deaminase to purine nucleoside phosphorylase gradually decreased from 21 in fraction 1 to 7 in the last fraction (blood T-cell value, 0.7). We conclude that this enzyme activity ratio is a useful marker for intrathymic T-cell maturation stages. In PHA-responsive cell fractions (3-6), the sensitivity to inhibition of the PHA response by (deoxy)adenosine and deoxyguanosine was inversely related to the enzyme activity ratio of ecto-5'-nucleotidase to deoxycytidine kinase. These findings are compatible with the hypothesis that intracellular concentrations of phosphorylated (deoxy)nucleosides are related to this inhibition. We conclude that the differences in purine metabolism among the various (mitogen-responsive) human thymocyte fractions are related to lymphoid cell function. Since the number of cells contributing to the enzyme activities and the number of cells contributing to the proliferative response (about 15% of unseparated cells) differ considerably, it is not possible to evaluate enzyme activities in unseparated thymocytes in terms of relationships between purine metabolism and lymphocyte function.