The neuropeptide hypocretin, also known as orexin, has been implicated in waking since its deletion leads to the sleep disorder narcolepsy. Hypocretin neurons project to major arousal areas, and in an effort to determine which region is responsible for the changes in sleep-wake architecture we have developed the neurotoxin hypocretin2-saporin, which lesions hypocretin receptor bearing neurons. Here, in rats, we investigate the effects of hypocretin2-saporin lesions of the substantia nigra and ventral tegmental area in the regulation of sleep and wakefulness. Bilateral injection of hypocretin2-sap into both the ventral tegmental area and substantia nigra (92 and 184 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra) or into the substantia nigra alone (184 ng/microl, 0.5 microl) produced insomnia. The insomnia seemed to be associated with a large increase in locomotion on days 4 and 6 postinjection, as hyperactivity and stereotypic movements were consistently observed on the video recordings in all lesioned rats. In these rats, a nearly complete loss of both tyrosine hydroxylase and neuron-specific nuclear protein (neuronal nuclei) immunoreactive cells in the substantia nigra as well as diminution of tyrosine hydroxylase-immunoreactive fibers in the caudate putamen was found. Following bilateral injection of hypocretin2-sap at a lower concentration (46 ng/microl, 0.25 microl in the ventral tegmental area and 0.5 microl in the substantia nigra), very little reduction in the number of tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons and only a temporary increase in wakefulness (17.4% increase during light-off period on day 6 postinjection) were observed. Ventral tegmental area lesions (184 ng/mul of hypocretin2-sap, 0.25 microl, bilateral injections) did not produce significant changes in sleep, although most of the tyrosine hydroxylase- and neuronal nuclei-immunoreactive neurons in the ventral tegmental area were destroyed. Insomnia following hypocretin2-sap lesions of the substantia nigra could be secondary to increased motor activity resulting from reduction of tonic inhibitory control by the substantia nigra.