Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase which in vitro adds deoxynucleotides to the 3' OH end of a primer without a template. This function and its distribution in bone marrow pre-T cells and thymocytes suggest a role for this enzyme in the development of the immune system. We measured TdT activity in thymus biopsy specimens of patients with immunodeficiency disease to gain possible insight into the biology of this enzyme and/or the pathogenesis of immune deficient states. Thymuses from four patients with severe combined immunodeficiency disease (SCID) and one with DiGeorge syndrome were assayed for enzyme activity with and without ATP, a specific inhibitor of TdT. In two patients with SCID, specific enzyme activity (pmol/0.5 g tissue) was 50 and 157%, respectively, of age and sex-matched controls; one patient had no enzyme activity. TdT activity in the only adenosine deaminase deficient patient was 23% and was not inhibited by ATP. Crude extract from the DiGeorge thymus had 42% activity, but was not tested with ATP. Purification of crude extracts by DEAE, phosphocellulose and oligodT chromatography was performed to determine if specific enzyme activity had been masked. After phosphocellulose chromatography, ATP noninhibitable activity was removed from one fraction pool in one patient, but not from a second pool. Two other patients had TdT activity in low salt eluates (0.05 M KCl) rather than where expected. Purification of these samples by oligodT chromatography resulted in specific enzyme activity comparable to age-matched controls. The known inhibition of TdT activity by ATP suggests that the nucleotides and deoxynucleotides which accumulate in lymphocytes in SCID may account for the abnormal TdT activity seen in these patient specimens. Examination of TdT activity in immunodeficient patients may allow delineation of this enzyme's biological function.