The behavioral effects of carbachol microinjection into the pontine, midbrain and medullary reticular formation (RF) have been studied. Enhancement of electrographic desynchronized (D) sleep signs and D sleep-like behavior was specific to the pons; these parameters were contrastingly suppressed following cholinergic stimulation of the midbrain and medullary RF. Polygraphically defined wakefulness (W) was increased from the midbrain and medulla and was characterized by stereotyped motor activity, whereas slow wave (S) sleep was reduced or eliminated when carbachol was administered into all 3 brainstem regions. In addition, as a function of the site of carbachol administration within the pons, some qualitative electrographic differences between carbachol-induced D sleep and the physiological D sleep state were observed. These results are consistent with the hypotheses that the pontine RF is involved in the cholinergic initiation and/or maintenance of D sleep and that the midbrain RF mediates arousal via cholinoceptive reticular cells. The hypothesis that the medullary RF plays a major role in the generation of S sleep and the atonia of D sleep is not supported by these data, but non-cholinergic mechanisms of these proposed roles remain to be investigated. Preliminary reports of these data have been published.