Adenosine is a potent regulator of acetylcholine release in the striatum, yet the mechanisms mediating this regulation are largely undefined. To begin to fill this gap, adenosine receptor expression and coupling to voltage-dependent Ca(2+) channels were studied in cholinergic interneurons by combined whole cell voltage-clamp recording and single-cell reverse transcription-polymerase chain reaction. Cholinergic interneurons were identified by the presence of choline acetyltransferase mRNA. Nearly all of these interneurons (90%, n = 28) expressed detectable levels of A(1) adenosine receptor mRNA. A(2a) and A(2b) receptor mRNAs were less frequently detected. A(3) receptor mRNA was undetectable. Adenosine rapidly and reversibly reduced N-type Ca(2+) currents in cholinergic interneurons. The A(1) receptor antagonist 8-cyclopentyl-1, 3-dimethylxanthine completely blocked the effect of adenosine. The IC(50) of the A(1) receptor selective agonist 2-chloro-N6-cyclopentyladenosine was 45 nM, whereas it was near 30 microM for the A(2a) receptor agonist CGS-21680. Dialysis with GDPbetaS or brief exposure to the G protein (G(i/o)) alkylating agent N-ethylmaleimide also blocked the adenosine modulation. The reduction in N-type currents was partially reversed by depolarizing prepulses. A membrane-delimited pathway mediated the modulation, because it was not seen in cell-attached patches when agonist was applied to the bath. Activation of protein kinase C attenuated the adenosine modulation. Taken together, our results argue that activation of A(1) adenosine receptors in cholinergic interneurons reduces N-type Ca(2+) currents via a membrane-delimited, G(i/o) class G-protein pathway that is regulated by protein kinase C. These observations establish a cellular mechanism by which adenosine may serve to reduce acetylcholine release.