Various fluorescent nucleoside agonists of the A 3 adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N 6 position and rigid A 3 AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA 3 AR expressed in whole CHO cells and membranes. By FCM, binding was A 3 AR-mediated (blocked by A 3 AR antagonist, at least half through internalization), with t 1/2 for association 38min in mA 3 AR-HEK293 cells; 26.4min in sucrose-treated hA 3 AR-CHO cells (K d 31nM). Membrane binding indicated moderate mA 3 AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA 3 AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A 3 AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A 3 AR.