Abstract Spontaneous proliferation of thymocytes after 20–25 h of culture was significantly increased by the presence of adenosine deaminase (ADA) or theophylline. The effect of ADA was counteracted by the ADA inhibitor EHNA. When given alone, EHNA inhibited proliferation. This effect was not blocked by inhibition of adenosine uptake with dipyridamol. These results suggest that proliferation in culture is regulated by a balance between endogenous adenosine and ADA, controlling the influence of adenosine on the intracellular cyclic AMP level via an adenosine receptor on the surface of thymocytes. According to the hypothesis, ADA would stimulate proliferation by decreasing extracellular adenosine levels and theophylline by blocking adenosine receptors on thymocytes. EHNA would inhibit proliferation by increasing extracellular adenosine levels. In accordance with this interpretation, the adenosine analogue phenylisopropyl adenosine (PIA) inhibited proliferation and the effect could be inhibited by theophylline. The postulated effect of endogenous adenosine could not be mimicked by a single administration of exogenous adenosine. Whereas most doses of adenosine were without effect, a high dose of adenosine (0.1 mM) in combination with EHNA unexpectedly stimulated proliferation. Since the effect was blocked by dipyridamol, an intracellular site of action for adenosine is suggested in this case.