Recombinant human A_2B adenosine receptors (A_2BARs) and receptors extended on the amino terminus with hexahistidine and the FLAG epitope, DYKDDDDK (H/F-A_2B) were stably overexpressed (to >20,000 fmol/mg protein) in human embryonic kidney 293 cells (HEK-A_2B). By Western blotting, the H/F-A_2Breceptor runs as a 34.8-kDa glycoprotein. Pharmacological properties of A_2BARs were characterized with¹²⁵I-3-aminobenzyl-8-phenyl-(4-oxyacetic acid)-1-propylxanthine (K _D, 36 nM). In competition binding assays, the affinity of agonists is reduced by substitution on either the N ⁶- or the C-2 position of the adenine ring, whereas 5′-substitutions increase affinity, resulting in the potency order: 5′-N-ethylcarboxamidoadenosine (NECA) ≫N ⁶-aminobenzyl-NECA ≈2-chloroadenosine > 2-[4-(2-carboxyethyl)phenethylamino]-NECA (CGS21680) >N ⁶-aminobenzyladenosine. The A_2BAR is potently blocked by the A_2A-selective antagonist 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo-[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol (ZM241385; K _I, 32 nM for A_2B, 1.4 nM for A_2A) and the A₁ selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (K _I, 50.5 nM for A_2B; 2.5 nM for A₁). TheK _I values for the antiasthmatic xanthines, theophylline (7.8 μM) and enprofylline (6.4 μM), are below their therapeutic plasma concentrations (20 to 50 μM), and agree withK _I determinations for inhibition of NECA-stimulated cAMP accumulation in HEK-A_2B cells. NECA orN ⁶-(2-iodo)benzyl-5′-N-methylcarboxamidodoadenosine (IB-MECA) stimulate inositol trisphosphates and calcium accumulation in HEK-A_2B or HEK-A₃ cells, respectively, but only the A₃ response is prevented by pertussis toxin. In human HMC-1 mast cells, A_2BAR activation stimulates calcium mobilization and cAMP accumulation. We conclude that HEK-A_2B cells and HMC-1 mast cells possess A_2BAR glycoproteins that are coupled to both G_q/11 and G_s.