The objective of the present study was to characterize the morphology of serotoninergic axons in cerebral cortex of the rat and to determine whether dissimilar axon terminals arise from the dorsal vs. the median raphe nuclei. The anterograde tracer PHA-L was administered by iontophoresis into the dorsal (DR) and median (MR) raphe nuclei, and the morphologic features of the respective axonal projections from raphe to forebrain were analyzed. We have observed consistent structural differences between the axons from these two nuclei. Anterogradely labeled axons which arise from cells in the MR are characterized by large, spherical varicosities (type M axons) and by variations in axonal diameter. In contrast, DR fibers are very fine and typically have small, pleomorphic varicosities that are granular or fusiform in shape (type D axons). Similar features of serotonin (5-HT) axon morphology are also evident in 5-HT immunocytochemical preparations. In addition to structural differences, there is a differential topographic distribution of MR vs. DR fibers, with MR axons concentrated in particular areas of limbic cortex such as dentate gyrus, posterior cingulate, and entorhinal areas as well as in parietal cortex. Immunofluorescence with dual labels shows that over two-thirds of the raphe-cortical axons are serotoninergic. The dissimilarities in axon morphology indicate that individual raphe nuclei may form different patterns of synaptic organization. Based on the evidence that the dorsal and median raphe nuclei give rise to morphologically different axon terminals, we conclude that 5-HT axons in cortex may be subdivided into two distinct projections. This proposal is in accord with other, recent data showing that the two axon types have different pharmacologic properties and are likely to be functionally different.