Adenosine has been proposed as a key factor regulating the metabolic balance between energy supply and demand in the central nervous system. Because astrocytes represent an important cellular element in the control of brain energy metabolism, we investigated whether adenosine could induce long-term changes of glycogen levels in primary cultures of mouse cortical astrocytes. We observed that adenosine increased glycogen content, up to 300%, in a time- (maximum at 8 h) and concentration-dependent manner with an EC₅₀ of 9.69 µM. Pharmacological experiments using the broad-spectrum agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and specific agonists for the A₁, A_2A, and A₃ receptors [N⁶-cyclopentyladenosine (CPA), CGS-21680, and IB-MECA, respectively] suggest that the effect of adenosine is mediated through activation of the low-affinity A_2B adenosine receptor subtype. Interestingly, adenosine induces in parallel the expression of the protein targeting to glycogen (PTG), one of the protein phosphatase-1 glycogen-targeting subunits that has been implicated in the control of glycogen levels in various tissues. These results indicate that adenosine can exert long-term control over glycogen levels in astrocytes and might therefore play a significant role in physiological and/or pathological processes involving long-term modulation of brain energy metabolism.