Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT₁ MF-2 smooth muscle cells were incubated with 10 μM [³H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (IAA, 5 mM) increased tritium release by 63% (P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 μM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [³H]adenosine. The adenosine A₁ receptor selective agonist N⁶-cyclohexyladenosine (CHA, 300 nM) increased the release of [³H]purine nucleoside induced by IAA treatment by 39% (P < 0.05). This increase was blocked by the A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 μM). Treatment of cells with UTP (100 μM), histamine (100 μM), or phorbol-12-myristate-13-acetate (PMA, 10 μM) also increased [³H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [³H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A₁ receptor activation increased nucleoside efflux from metabolically stressed DDT₁ MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.