Cyclic 3′,5′-adenosine monophosphate (cAMP) is secreted as the chemotactic signal by aggregating amoebae of the cellular slime mold Dictyostelium discoideum. We have used ultramicrotechniques in the biochemical analysis of cyclic nucleotide phosphodiesterase (PD) distribution in individual aggregates at various stages of development. With handmade constriction pipettes in microliter volumes, sections of lyophilized individuals weighing 20–100 ng could be assayed in a reaction coupled to 5′-nucleotidase. Phosphodiesterase activity was measured at pH 7.5 with 12 μM cAMP. cAMP-PD activity in aggregates ranged from 20–40 mmol/h/kg. In the pseudoplasmodium it had dropped to 5–10 mmol/h/kg and a difference in activity between the anterior prestalk cells and posterior prespore cells began to appear. The utmost posterior sections showed elevated phosphodiesterase from this stage onward. During culmination, activity rose to 40–60 mmol/h/kg associated with the developing stalk, while it declined in the spore mass. The papilla remained constant at 5–10 mmol/h/kg. The pattern of localization in the stalk was the same when cGMP was used as substrate. Extracellular phosphodiesterase inhibitor produced at the aggregation stage was found to reduce the localized activity in the culmination stage by 50–80%, with the most marked inhibition occurring in the center of the papilla. We found no evidence of endogenous heat-stable phosphodiesterase inhibitor within the culminating sorocarp.