Endogenous adenosine plays a crucial role in maintaining energy homeostasis and adenosine levels are tightly regulated across neural circuits. In the dorsal medial striatum (DMS) adenosine inhibits neurotransmitter release, but the source and mechanism underlying its accumulation are largely unknown. Opioids also inhibit neurotransmitter release in the DMS and influence adenosine accumulation after prolonged exposure. However, how these two neurotransmitter systems interact acutely is also largely unknown. This study demonstrates that activation of μ opioid receptors (MORs), but not δ opioid receptors (DORs) or κ opioid receptors (KORs), inhibits tonic activation of adenosine A1Rs via a cyclic adenosine monophosphate (cAMP) dependent mechanism in both male and female mice. Further, selectively knocking-out MORs from thalamic presynaptic terminals and postsynaptic medium spiny neurons (MSNs) revealed that activation of MORs on D1R positive MSNs, but not D2R positive MSNs, is necessary to inhibit tonic adeno...