In Dictyostelium discoideum, extracellular cAMP activates adenylate cyclase, which leads to an increase in intracellular cAMP and the rate of cAMP secretion. The signaling response to a constant cAMP stimulus is terminated after several minutes by an adaptation mechanism. The time-course of adaptation to stimuli of 10-6 or 10-7 M cAMP was assessed. We used a perfusion technique to deliver defined cAMP stimuli to [3H]adenosine-labeled amoebae and monitored their secretion of [3H]cAMP. Amoebae were pretreated with 10-6 or lO-7 M cAMP for periods of 0.33-12 minutes, and then immediately given test stimuli of 10-8 M to 2.5 × 10-7 M cAMP. The response to a given test stimulus was progressively attenuated and finally extinguished as the duration of the pretreatment stimulus increased. During pretreatment with 10-6 M cAMP, the rates of attenuation could be ranked according to the concentration of the test stimulus. The responses to test stimuli of 10-8, 5 × 10-8, 10-7, or 2.5 × 10-8 M cAMP were extinguished after ≈1, 2.25, 2.5, and 10 min, respectively. 1.5 min of stimulation with 10-6 M cAMP was necessary to extinguish the response of a test stimulus of 10-7 M cAMP. Our data suggest that adaptation begins within 20 s of stimulation, rises rapidly for ≈2.5 min, and reaches a plateau after ≈10 min. The absolute rate of rise was faster during pretreatment with 10-8 than with 10-8 M cAMP. These results support a working hypothesis in which the occupancy of surface cAMP receptors leads to changes in two opposing cellular processes, excitation and adaptation, that control the activity of D. discoideum adenylate cyclase.