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Background Adenosine serves many functions within the CNS, including inhibitory and excitatory control of neurotransmission. The understanding of adenosine dynamics in the brain is of fundamental importance. The goal of the present study was to explore subsecond adenosine fluctuations in the rat brain in vivo . Method Long Evans rats were anesthetized and a carbon fiber electrode was positioned in the motor cortex or dorsal striatum. Real time electrochemical recordings were made at the carbon fiber electrodes every 100 ms by applying a triangular waveform (−0.4 to +1.5 V, 400 V/s). Adenosine spikes were identified by the background-subtracted cyclic voltammogram. Results The frequency of detected adenosine spikes was relatively stable in both tested regions, and the time intervals between spikes were regular and lasted from 1 to 5 s within an animal. Spike frequency ranged from 0.5 to 1.5 Hz in both the motor cortex and the dorsal striatum. Average spike amplitudes were 85 ± 11 and 66 ± 7 nM for the motor cortex and the dorsal striatum, respectively. Comparison with existing methods The current study established that adenosine signaling can operate on a fast time scale (within seconds) to modulate brain functions. Conclusions This finding suggests that spontaneous adenosine release may play a fast, dynamic role in regulating an organism's response to external events. Therefore, adenosine transmission in the brain may have characteristics similar to those of classical neurotransmitters, such as dopamine and norepinephrine.