The dopamine transporter (DAT) critically regulates the duration of the cellular actions of dopamine and the extent to which dopamine diffuses in the extracellular space. We sought to determine whether the reportedly greater diffusion of dopamine in the rat prefrontal cortex (PFC) as compared with the striatum is associated with a more restricted axonal distribution of the cortical DAT protein. By light microscopy, avidin-biotin-peroxidase immunostaining for DAT was visualized in fibers that were densely distributed within the dorsolateral striatum and the superficial layers of the dorsal anterior cingulate cortex. In contrast, DAT-labeled axons were distributed only sparsely to the deep layers of the prelimbic cortex. By electron microscopy, DAT-immunoreactive profiles in the striatum and cingulate cortex included both varicose and intervaricose segments of axons. However, DAT-labeled processes in the prelimbic cortex were almost exclusively intervaricose axon segments. Immunolabeling for tyrosine hydroxylase in adjacent sections of the prelimbic cortex was localized to both varicosities and intervaricose segments of axons. These qualitative observations were supported by a quantitative assessment in which the diameter of immunoreactive profiles was used as a relative measure of whether varicose or intervaricose axon segments were labeled. These results suggest that considerable extracellular diffusion of dopamine in the prelimbic PFC may result, at least in part, from a paucity of DAT content in mesocortical dopamine axons, as well as a distribution of the DAT protein at a distance from synaptic release sites. The results further suggest that different populations of dopamine neurons selectively target the DAT to different subcellular locations.