The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3':5'-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly within 1 min and was maximal by 10 to 20 min with approx. 2 and 10 muM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C]adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.