Four cyclic AMP-specific, rolipram-inhibited phosphodiesterases (PDE4s) have been identified in mammals; all four are homologs of dunce, a gene required for learning and memory in Drosophila. To determine the distribution of PDE4s in the mammalian brain, specific antibodies were generated against the proteins encoded by each of three dunce homologs PDE4A, PDE4B, and PDE4D in the mouse. On Western blots, these antibodies recognized multiple protein species in all brain regions studied. Immunohistochemical studies showed that both cell bodies and neuropil were well labeled in selected regions throughout the brain. Immunoreactivity for PDE4A was found predominantly in the anterior olfactory nucleus, subiculum, layer V pyramidal neurons from the cerebral cortex, and corticospinal tracts. By contrast, anti-PDE4B-labeled neurons were observed in the inferior olive, the paraventricular and supraoptic nuclei of the hypothalamus, and in the ventral striatum. Regions of neuropil containing high levels of PDE4B immunoreactivity included the cerebellar molecular layer, globus pallidus, nucleus accumbens, and substantia nigra. Anti-PDE4D antibody distinctly labeled cerebellar Purkinje cells as well as neurons in the medial habenula and thalamic nuclei. Fibers in the fasciculus retroflexus, interpeduncular nuclei, and periaqueductal gray were also stained with this antibody. These findings indicate that the distribution of PDE4s in the brain is remarkably segregated, and suggest that each of these enzymes has a unique functional role. Furthermore, the data support the notion that rolipram, the PDE4-specific inhibitor that acts as an antidepressant in humans, may mediate its behavioral effects through PDE4B, which is highly localized to neural pathways known to underlie reward and affect in mammals.