Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) and is accompanied by T-cell depletion and accumulation of both intracellular and extracellular adenosine (extAdo) and deoxyadenosine. To better understand the causes of T-cell depletion in vivo and to discriminate between extracellular and intracellular effects of exogenously added adenosine in vitro, we investigated mechanisms of 2 different effects of adenosine on murine thymocytes. These effects of adenosine include direct induction of apoptosis in about 6% to 15% thymocytes and inhibition of T-cell receptor (TCR)-induced activation of the majority of thymocytes with inhibited ADA. A_2A adenosine receptors, but not A_2B, A₁, or A₃ receptors, are shown to be mostly responsible for extAdo-triggered signaling (cyclic adenosine monophosphate [cAMP] accumulation) in murine thymocytes and this prompted studies of the effects of extAdo on thymocytes from A_2AR gene-deficient mice. It is found that direct apoptotic effects of extAdo on CD4⁺CD8⁺ double positive (DP) thymocytes are completely accounted for by signaling through A_2AR, with no contribution of intracellular lymphotoxicity or of compensating A_2BRs because only A_2AR +/+, but not A_2AR −/− thymocytes were susceptible to apoptotic effects of extAdo. Studies of the effects of cAMP-raising agents support observations of extAdo/A_2AR/cAMP–triggered apoptosis in DP thymocytes. Unexpectedly, the extAdo strongly inhibited TCR-triggered activation of both A_2AR +/+ and A_2AR −/− thymocytes in the presence of ADA inhibitors. This was confirmed with thymocytes from ADA gene-deficient mice, suggesting the existence of A_2AR-independent effects of extAdo on thymocytes. The presented data raises questions about the identity and functional role of A_2AR-expressing thymocytes in T-cell differentiation and of the role of TCR-antagonizing effects of extAdo in conditions of ADA SCID.