Adenosine, an endogenous modulator of synaptic transmission, has been implicated in regulation of sleep and arousal. The effect of adenosine on neuronal excitability depends on its concentration in the extracellular space. The present study shows that the state of activity of laboratory rats determines the level of tonic inhibition by adenosine in hippocampal slices prepared from these animals. Thus, slices taken at the end of the active period showed significantly more inhibition by adenosine, as determined by the effects of the A1 receptor blocker 8-CPT, in comparison to slices taken in the inactive state. The results support the proposed role of adenosine in regulation of sleep and arousal and point to the importance of the time of day at which slices for electrophysiological experiments are prepared.