In order to determine the cholinoceptive brainstem structures critical for PS generation, we investigated the effect on PS induction of the injection of a small dose and volume (0.4 microgram/0.2 microliter) of the cholinergic agonist carbachol in the following caudal brainstem structures: 1) the caudal mesencephalic reticular formation, especially the nucleus pedunculopontinus pars compacta or X area; 2) the mediodorsal pontine tegmentum, in particular the nuclei locus coeruleus (LC), locus coeruleus alpha (LC alpha), peri-locus coeruleus alpha (peri-LC alpha) and laterodorsalis tegmenti (Ldt); 3) the pontine; and 4) bulbar gigantocellular (FTG) and magnocellular tegmental fields (FTM). We found that the only brainstem area from which a high amount of PS was induced by carbachol applications with short latencies, less than 5 minutes, is the mediodorsal pontine tegumentum, namely the LC alpha and peri-LC alpha, where ChAT-and TH- immunoreactive neurons are intermingled. Injections in an area immediately ventral to the peri-LC alpha induced physiological states resembling PS but lacking certain electrophysiological (PS-like) and behavioral components of PS (dissociated states I and II). The weak PS induction following carbachol administration in the anteromedial part of the FTG was due to the spread of the drug toward the efficient site since the latencies to PS onset were in the range of 20 to 60 minutes. No effects on PS generation were obtained after carbachol microinjections in the LC and the laterocaudal part of the FTG, while carbachol injections in the X area or in the bulbar FTG or FTM resulted in the increase of waking and the decrease of PS. In addition to these effects on PS induction, we also found that carbachol induced: 1) stereotyped PGO-like bursts when injected in the ventral part of the FTG and the rostral part of the FTM, 2) postural atonia with very short latencies, less than two minutes, when injected in the LC alpha and peri-LC alpha; and 3) hippocampal theta waves of 3-5 Hz persisting during light slow wave sleep (S1) when injected in and around the LC alpha and peri-LC alpha and in some points of the mediocaudal part of the FTG. These results support the hypothesis that PS is generated by highly localized neuronal populations and suggest that the mediodorsal pontine tegmentum (namely the nuclei LC alpha and peri-LC alpha) may represent a cholinoceptive PS generator.