Chronic exposure to caffeine increases the number of adenosine receptors (up-regulation) but these observations have been mostly limited to animal models that study A1 adenosine receptors. The regulation of adenosine receptors by caffeine in humans and, in particular A2 receptors, remains largely unexplored. The purpose of this study was to test the hypothesis that withdrawal from chronic caffeine administration results in up-regulation of A2 adenosine receptors in humans. The authors also wanted to determine whether caffeine induces homologous or heterologous up-regulation. Caffeine 250 mg three times daily was given orally to a total of 19 normal volunteers for 7 days. Platelets were obtained at base line and 12 and 60 hr after the last dose of caffeine and the antiaggregation responses to adenosine and prostacyclin receptors were evaluated ex vivo. Plasma caffeine levels remained elevated at 22 microM 12 hr after the last dose but decreased to 0.6 microM at 60 hr. Adenosine receptor activation with the agonist 5'-N-ethylcarboxamidoadenosine and prostacyclin receptor activation with iloprost or prostaglandin E1 produced a greater antiaggregation effect at 60 hr postcaffeine. Increased responsiveness to both receptors could also be demonstrated at 12 hr after removal of caffeine by washing the platelets. Sensitization to the actions of prostacyclin, however, was reversed if caffeine was added ex vivo. These results support the hypothesis that chronic caffeine exposure induces heterologous up-regulation of adenosine and prostacyclin receptors in humans and implies that endogenous adenosine normally modulates platelet adenosine receptors in vivo. These findings may be relevant to the caffeine withdrawal syndrome observed in humans.