Adenosine-induced coronary vasodilation is predominantly A(2A) adenosine receptor (AR)-mediated, whereas A(1) AR is known to negatively modulate the coronary flow (CF). However, the coronary responses to adenosine in hyperlipidemia and atherosclerosis are not well understood. Using hyperlipidemic/atherosclerotic apolipoprotein E (APOE)-knockout mice, CF responses to nonspecific adenosine agonist (5'-N-ethylcarboxamide adenosine, NECA) and specific adenosine agonists (2-chloro-N6-cyclopentyl-adenosine [CCPA, A(1) AR-specific] and CGS-21680, A(2A) AR-specific) were assessed using isolated Langendorff hearts. Western blot analysis was performed in the aorta from APOE and their wild-type (WT) control (C57BL/6J). Baseline CF (expressed as mL/min/g heart weight) was not different among WT (13.23 ± 3.58), APOE (13.22 ± 2.78), and APOE on high-fat diet (HFD) for 12 weeks (APOE-HFD, 12.37 ± 4.76). Concentration response curves induced by CGS-21680 were significantly shifted to the left in APOE and APOE-HFD when compared with WT. CCPA induced an increase in CF only at 10(-6) M in all groups and the effect was reversed by the addition of a selective A(2A) AR antagonist, SCH-58261 (10(-6) M), and a significant decrease in CF from baseline was observed. Western blot analysis showed a significant upregulation of A(2A) AR in the aorta from APOE and APOE-HFD. This study provides the first evidence that CF responses to A(2A) AR stimulation were upregulated in hyperlipidemic/atherosclerotic animals. The speculation is that the use of A(2A) AR-specific agonist for myocardial perfusion imaging (such as regadenoson) could overestimate the coronary reserve in coronary artery disease patients.