A detailed characterization of the cyclic nucleotide phosphodiesterase (PDEs) from normal Drosophila melanogaster was made, including purification of the two major enzymes to near homogeneity. A third more labile phosphodiesterase also was identified in crude homogenates. The total activity per fly of one of these three enzymes, PDE-II, is strongly influenced by the dunce locus. Two independently derived dunce mutants produce variations of PDE-II with modified intrinsic properties: a marked decrease of thermal stability in dunce and a 10- fold increase in the Michaelis kinetic constant in dunce. These defects, which persisted in purified preparations of PDE-II, were mapped genetically to dunce. The results support the identification of dunce as the structural locus for PDE-II. The tight connection between the dunce gene and the PDE-II enzyme indicates that defective cyclic adenosine 3′:5′-monophosphate metabolism is the primary lesion which leads to failure of dunce flies to learn in the olfactory associative conditioning paradigm of Quinn et al. (Quinn, W. G., W. A. Harris, and S. Benzer (1974) Proct. Natl. Acad. Sci. U. S. A. 71: 708–712).