We investigated whether A₃ adenosine receptor (A₃AR) is involved in endothelium mediated contraction through cyclooxygenases (COXs) with the use of wild type (WT) and A₃ knock-out (A₃KO) mice aorta. A₃AR selective agonist, Cl-IBMECA produced a concentration-dependent contraction (EC₅₀: 2.9±0.2x10^-9 M) in WT mouse aorta with intact endothelium (+E), and negligible effects in A₃KO (+E) aorta. At 10^-7 M, contractions produced by Cl-IBMECA were 29% in WT (+E), while being insignificant in A₃KO (+E) aorta. Cl-IBMECA-induced responses were abolished in endothelium denuded tissues (-E) both in WT and A₃KO aorta. A₃AR gene and protein expression were reduced by 74% and 72% (p<0.05), respectively in WT (-E) as compared to WT (+E) aorta while being undetected in A₃KO (+E/-E) aorta. Indomethacin (non-specific COXs blocker, 10^-5 M), SC-560 (specific COX-1 blocker, 10^-8 M), SQ 29549 [thromboxane prostanoid receptor (TP) antagonist (10^-6 M)] and furegrelate [thromboxane synthase (TS) inhibitor, 10^-5 M] inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced TXB₂ production was attenuated significantly by indomethacin, SC-560 and furegrelate in WT (+E) aorta while having negligible effects in A₃KO (+E) aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA induced contraction both in WT (+E) and A₃KO (+E) aorta. Cl-IBMECA-induced increase in COX-1 and TP receptor expression was significantly inhibited by MRS1523, a specific A₃AR antagonist in WT (+E) aorta. Expression of both A₃AR and COX-1 was located mostly on endothelium of WT and A₃KO (+E) aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A₃AR- mediated contraction via endothelium.