Chronic cannabinoid treatment results in the development of tolerance. Adenylyl cyclase superactivation, induced by chronic cannabinoid agonist administration, is regarded as one of the molecular mechanisms leading to tolerance. In the present study, the effect of cyclosporine on adenylyl cyclase superactivation after chronic exposure to WIN 55,212–2, a cannabinoid receptor agonist, was studied. Chronic treatment (18 h) with WIN 55,212–2 induced a significant increase in cAMP levels in human astrocytoma cells (adenylyl cyclase superactivation). Acute treatment with cyclosporine (10 min) did not have any effect on WIN 55,212–2-induced adenylyl cyclase superactivation. But, chronic cyclosporine treatment (18 h), with concentration from 1 nM to 1 μM, attenuates the development of adenylyl cyclase superactivation after chronic WIN 55,212–2 treatment. Our findings show that cyclosporine attenuates chronic cannabinoid-mediated adenylyl cyclase superactivation.