In Dictyostelium discoideum, binding of cAMP to high affinity surface receptors leads to a rapid activation of adenylate cyclase followed by subsequent adaptation within several minutes. The rate of secretion of [3H]cAMP, which reflects the state of activation of the enzyme, was measured. Caffeine noncompetitively inhibited the response to cAMP. Inhibition was rapidly reversible and pretreatment of cells with caffeine for up to 22 min had little effect on the subsequent responsiveness to cAMP. However, cells pretreated with caffeine plus cAMP for ≥ 8 min did not respond when caffeine was removed and the same concentration of cAMP was applied. The following observations indicate that both adaptation and deaptation to cAMP occurred to the same extent and at the same rate whether or not cAMP synthesis was inhibited. First, when cells were pretreated with 10-9-10-6 M cAMP in the presence or absence of caffeine and the stimulus was switched to a saturating dose of cAMP, the response to the increment was the same whether or not the initial response was blocked. Second, cells progressively lost responsiveness to 10-6 M cAMP as pretreatment with 10-6 M cAMP plus caffeine was extended from 0 to 8 min with the same time course as for those pretreated with 10-6 M cAMP alone. Third, cells which were adapted in the presence of caffeine and cAMP deadapted within the same time period as controls when cAMP was removed. These observations demonstrate that while some part of the activation process is inhibited by caffeine the adaptation process is unaffected. Our conclusion is that adaptation does not depend on the activation of adenylate cyclase.