Previous studies have shown that there are morphologically dissimilar serotonergic (5-HT) axon types in rat cerebral cortex which are differentially sensitive to the neurotoxic effects of certain psychotropic drugs: methylenedioxyamphetamines (MDA and MDMA) and p-chloroamphetamine (PCA) cause degeneration of fine 5-HT axon terminals in cortex, while sparing beaded axons. Moreover, a recent anterograde transport study suggests that fine and beaded 5-HT axons arise from the dorsal raphe (DR) and median raphe (MR) nuclei, respectively. These data led us to propose that the DR projection to neocortex is selectively vulnerable to the neurotoxic effects of PCA, while the MR projection is resistant; this hypothesis was tested in the present study by comparing retrograde axonal transport of the fluorescent tracer Fluoro-Gold in PCA-treated and control rats. Using this method, only axons that survive PCA treatment can take up and transport the injected label back to the cell bodies of origin, thus allowing us to determine which raphe-cortical projections remain intact after PCA. The results show that PCA administration produces a loss of fine 5-HT axon terminals in neocortex and a concomitant reduction in the number of retrogradely labeled neurons in the DR (77% decrease), when compared to controls. In contrast, beaded 5-HT axon terminals are spared and the number of labeled neurons in the MR remains unchanged after PCA. These results demonstrate that DR and MR projections to cortex are differentially vulnerable to PCA: fine axon terminals arise from neurons in the DR and are highly sensitive to the neurotoxic effects, whereas beaded axons from the MR are resistant. We therefore propose that there are two anatomically and functionally separate 5-HT projections to cortex having different (1) nuclei of origin, (2) axon morphology, (3) regional distributions, and (4) pharmacological properties. Since the mood-altering substances MDA, MDMA, and PCA act specifically upon 5-HT axon terminals from the dorsal raphe nucleus, DR neurons may be preferentially involved in the control of affective state.