Caffeine is believed to act by blocking adenosine A₁and A_2Areceptors (A₁R, A_2AR), indicating that some A₁receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A₁R (A₁R^−/−). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A₁R^+/+mice, but A₁R^−/−mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A₁R^−/−mice. In A₁R^+/−mice the potency of adenosine was halved, as was the number of A₁R. In A₁R^−/−mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A₁Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important.