A simple assay has been developed to measure cGMP-specific phosphodiesterase (cGPD) activity in crude soluble extracts of amoebae of Dictyostelium discoideum. When amoebae of different wild-type strains were starved on buffered agar, all strains exhibited an 8- to 12-fold increase in cGMP-specific hydrolyzing activity during development, with the major increase occurring at aggregation. cGMP-specific activity was found in both prestalk and prespore cells. To determine if the elevated cGMP-specific hydrolyzing activity observed during late development was associated with the same enzyme present in vegetative cells, cGMP-specific activities were partially purified from cells at different developmental stages and characterized. Activity in vegetative cells was fractionated by gel filtration into three components with molecular weights of approximately 172,000, 115,000 and 56,000. In contrast, cells starved 4 hr in suspension or 18 hr on agar possessed only the 172,000 or 115,000 Mr forms, respectively. The low-molecular-weight enzyme differed from the two larger forms in kinetic properties and in sensitivity to sulfhydryl reagents. Nevertheless, the three activities probably represent different forms of the same enzyme because mutants defective at the stmF locus lacked appreciable cGMP-specific hydrolyzing activity throughout development. These results indicate that D. discoideum produces a single cGPD which is strongly developmentally regulated. These findings further suggest that intracellular cGMP might be involved in regulating postaggregative as well as preaggregative development.