Treatment with conventional antipsychotic drugs (APDs) is accompanied by extrapyramidal side effects (EPS), which are thought to be due to striatal dopamine D2 receptor blockade. In contrast, treatment with atypical APDs is marked by a low incidence or absence of EPS. The reduced motor side effect liability of atypical APDs has been attributed to a high serotonin 5-HT2A receptor affinity coupled with a relatively low D2 affinity. Despite the high density of 5-HT2A binding sites in the striatum, there are few detectable 5-HT2A mRNA-expressing neurons in the striatum. This suggests that most striatal 5-HT2A receptors are heteroceptors located on afferent axons. A combined retrograde tracer-immunohistochemistry method was used to determine the sites of origin of striatal 5-HT2A-like immunoreactive axons. 5-HT2A-like immunoreactive neurons in both the cortex and globus pallidus were retrogradely labeled from the striatum; very few nigrostriatal or thalamostriatal neurons expressed 5-HT2A-like immunoreactivity. Within the striatum, parvalbumin-containing interneurons displayed 5-HT2A immunolabeling; these neurons are the targets of cortical and pallidal projections. Our data indicate that cortico- and pallido-striatal neurons are the major source of 5-HT2A receptor binding in the striatum, and suggest that cortico- and pallido-striatal neurons are strategically positioned to reduce the motor side effects that accompany striatal D2 receptor blockade or are seen in parkinsonism. Synapse 39:297–304, 2001. © 2001 Wiley-Liss, Inc.