Sleep and sleep intensity are enhanced by adenosine and its receptor agonists, whereas adenosine receptor antagonists induce wakefulness. Adenosine kinase (ADK) is the primary enzyme metabolizing adenosine in adult brain. To investigate whether adenosine metabolism or clearance affects sleep, we recorded sleep in mice with engineered mutations in Adk. Adk-tg mice overexpress a transgene encoding the cytoplasmic isoform of ADK in the brain but lack the nuclear isoform of the enzyme. Wild-type mice and Adk(+/-) mice that have a 50% reduction of the cytoplasmic and the nuclear isoforms of ADK served as controls. Adk-tg mice showed a remarkable reduction of EEG power in low frequencies in all vigilance states and in theta activity (6.25-11 Hz) in rapid eye movement (REM) sleep and waking. Adk-tg mice were awake 58 min more per day than wild-type mice and spent significantly less time in REM sleep (102 ± 3 vs 128 ± 3 min in wild type). After sleep deprivation, slow-wave activity (0.75-4 Hz), the intensity component of non-rapid eye movement sleep, increased significantly less in Adk-tg mice and their slow-wave energy was reduced. In contrast, the vigilance states and EEG spectra of Adk(+/-) and wild-type mice did not differ. Our data suggest that overexpression of the cytoplasmic isoform of ADK is sufficient to alter sleep physiology. ADK might orchestrate neurotransmitter pathways involved in the generation of EEG oscillations and regulation of sleep.