Immunodeficient children who lack the purine metabolic enzyme adenosine deaminase have markedly elevated plasma concentrations of 2'-deoxyadenosine and adenosine. However, little information exists concerning the magnitude of endogenous 2'-deoxyadenosine and adenosine synthesis by normal human hematopoietic cells. In the present experiments, we have used the sensitive technique of high performance liquid chromatography to quantitate changes in 2'-deoxyadenosine and adenosine production during human lymphocyte mitogenesis. In the presence of the adenosine deaminase inhibitor deoxycoformycin, human T lymphocytes stimulated with phytohemagglutinin (PHA), and non-T lymphocytes stimulated with formalinized Staphylococcus aureus Cowan strain I, excreted 2'-deoxyadenosine into the cell medium. The nucleoside was detectable as early as 20 h after addition of mitogen. The time course of 2'-deoxyadenosine excretion correlated with the uptake of [methyl-3H]thymidine into nucleic acid. Mitogen-stimulated human lymphocytes produced only minimal amounts of adenosine. The results suggest that increased 2'-deoxyadenosine synthesis and release may normally accompany human lymphocyte mitogenesis.