Drosophila carrying the X-linked mutation dunce (dnc) showed poor learning in a negative reinforcement olfactory conditioning paradigm (Dudai, Y., Y.-N. Jan, D. Byers, W.G. Quinn, and S. Benzer (1976) Proc. Natl. Acad. Sci. U.S.A. 73: 1684-1688). More recently, dnc flies were shown to have reduced activity for one of two cAMP phosphodiesterases (PDEs) present in normal flies, PDE II, whereas PDE form I was unaffected (Byers, D., R. L. Davis, and J. A. Kiger, Jr. (1981) Nature 289: 79-81). A micro-assay technique is described that allows the separate measurement of PDE I and PDE II in crude extracts, based on specific inhibition of PDE I [3H]cAMP hydrolysis by cGMP. Using this technique, PDE II is shown to occur normally at high specific activity in the nervous system, consistent with the hypothesis that this enzyme plays a role in neuronal function. Reduced PDE II activity correlates with poor learning in dnc flies at three developmental stages (first and third instar larva and adult), as well as in response to genetic modification of dnc gene activity. Biochemical and genetic experiments fail to reveal any abnormal regulation of PDE II in dnc. The specific activity of PDE II is shown to correlate in a one to one fashion with the level of normal dnc gene (dnc+) activity at five different doses of dnc+. These results support the hypothesis that PDE II represents the primary product of the dnc gene, indicating a role for this enzyme in Drosophila learning.