The sesquiterpene endoperoxide antimalarial agents arteether and artemether have been reported to cause neurotoxicity with a discrete distribution in the brain stems of rats and dogs after multiple doses. The nature and distribution of the brain lesions suggest a specific neuronal target, the identity of which is unknown. In order to further investigate artemisinin analog-induced neurotoxicity, we evaluated several in vitro models: fetal rat primary neuronal cultures, fetal rat secondary astrocyte cultures, and transformed neuronal cultures (rat-derived neuroblastoma NG108-15 and mouse-derived neuroblastoma Neuro-2a). Results indicate that toxicity was specific for neuronal cell types but not glial cells. Neurotoxicity, as indexed by liberation of lactate dehydrogenase and/or inhibition of radiolabelled-leucine uptake, was seen in all three neuronal culture types, implicating a common target. In vitro neurotoxicity was dose and time dependent. Acute exposure to drug results in delayed, but not immediate, manifestations of cell toxicity. Structure-activity comparisons indicate that substitutions at positions 9 and 10 and stereoisomerism at position 10 of the artemisinin backbone influence the degree of toxicity. The endoperoxide is necessary but not sufficient for toxicity. Sodium artesunate and dihydroartemisinin, a metabolite common to all artemisinin analogs currently being developed for clinical use, are the most potent of all analogs tested. These results are consistent with a specific neuronal target, but the identity of the target(s) remains unknown.