Incubation of cerebral cortical slices of rat brain with 3H-adenosine in the presence of varying concentrations of morphine in vitro resulted in a dose-related increase in 3H-cAMP formation. In in vivo experiments, the rate of 3H-cAMP formation in cortical slices both 45 min and 4 h after the acute s.c. administration of a 10 mg/kg dose of morphine was significantly greater than that for saline-treated controls. A significant enhancement of cortical 3H-cAMP formation likewise became apparent 72 hr after s.c. implantation of two morphine pellets. This was not evident after only 24 h. In similar experiments undertaken with hypothalamic tissue, however, the rate of conversion of 3H-adenosine to 3H-cAMP remained similar to that of the controls. Administration of exogenous cAMP antagonized the analgesic response to morphine in both nontolerant and tolerant rats and accelerated the development of tolerance to morphine. These results may provide further evidence for a role of cAMP in the mediation of the central actions of morphine.