Objective: The aim was to detect cardiac A2 adenosine receptors through radioligand binding, and to assess the effect of ischaemia on these receptors. Methods: Isolated working rat hearts were subjected either to aerobic perfusion or to global ischaemia. A membrane fraction was prepared from ventricular tissue, and 3H-5'-N-ethylcarboxamide adenosine (NECA) binding was determined in the presence of N6-cyclopentyl adenosine (CPA). A2 binding was calculated as the fraction of NECA binding displaced by 100 μM CPA but not displaced by 50 nM CPA. Results: Analysis of A2 NECA binding according to single binding site model yielded Kd=22.0 nM, Bmax=34.0 fmol·mg−1 in control hearts; Kd=49.7 nM, Bmax=44.3 fmol·mg−1 in hearts subjected to 30 min ischaemia (p−1 for the high and low affinity sites respectively). The high affinity component of A2 NECA binding disappeared in the presence of the GTP analogue guanyl-5'-yl imidodiphosphate, suggesting the existence of multiple coupling states of the receptor. In the ischaemic group no significant improvement in data fitting was obtained with the two site model. Conclusions: The results provide evidence of the existence of cardiac A2 adenosine receptors. Ischaemia modifies receptor properties and appears to affect chiefly the high affinity component of A2 binding, possibly by preventing receptor interaction with membrane G proteins.