Previous data suggested that increases in extracellular adenosine in the basal forebrain mediated the sleep-inducing effects of prolonged wakefulness. The present study sought to determine if the state-related changes found in basal forebrain adenosine levels occurred uniformly throughout the brain. In vivo microdialysis sample collection coupled to microbore high-performance liquid chromatography measured extracellular adenosine levels in six brain regions of the cat: basal forebrain, cerebral cortex, thalamus, preoptic area of hypothalamus, dorsal raphe nucleus and pedunculopontine tegmental nucleus. In all these brain regions extracellular adenosine levels showed a similar decline of 15 to 20% during episodes of spontaneous sleep relative to wakefulness. Adenosine levels during non-rapid eye movement sleep did not differ from rapid eye movement sleep. In the course of 6h of sleep deprivation, adenosine levels increased significantly in the cholinergic region of the basal forebrain (to 140% of baseline) and, to a lesser extent in the cortex, but not in the other regions. Following sleep deprivation, basal forebrain adenosine levels declined very slowly, remaining significantly elevated throughout a 3-h period of recovery sleep, but elsewhere levels were either similar to, or lower than, baseline. The site-specific accumulation of adenosine during sleep deprivation suggests a differential regulation of adenosine levels by as yet unidentified mechanisms. Moreover, the unique pattern of sleep-related changes in basal forebrain adenosine level lends strong support to the hypothesis that the sleep-promoting effects of adenosine, as well as the sleepiness associated with prolonged wakefulness, are both mediated by adenosinergic inhibition of a cortically projecting basal forebrain arousal system.