We cloned and characterized the canine A₃ adenosine receptor (AR) and examined AR-induced degranulation of the BR line of canine mastocytoma cells. Canine A₃AR transcript is found predominantly in spleen, lung, liver, and testes and encodes a 314-amino acid heptahelical receptor.¹²⁵I-N ⁶-Aminobenzyladenosine binds to two affinity states of canine A₃AR withK _D values of 0.7 ± 0.1 and 16 ± 0.8 nm, reflecting G protein-coupled and -uncoupled receptors, respectively. Xanthine antagonists bind with similar affinities to human, canine, and rabbit receptors but with 80–400-fold lower affinities to rat A₃AR. Although canine BR mastocytoma cells contain A₁AR, A_2BAR, and A₃AR, degranulation seems to be mediated primarily by A_2BARs stimulated by the nonselective agonist 5′-N-ethylcarboxamidoadenosine (NECA) but not by the A₃-selective agonistN ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide. NECA-stimulated degranulation is not prevented by pertussis toxin and is blocked by enprofylline (K _i = 7 μm), an antiasthmatic xanthine with low affinity (K _i > 100 μm) for A₁AR, A_2AAR, and A₃AR. NECA increases canine mastocytoma cell cAMP, Ca²⁺, and inositol trisphosphate levels; these responses are antagonized half-maximally by 7–15 μm enprofylline. The results suggest that (i) the cloned canine A₃AR is structurally and pharmacologically more similar to human than to rat A₃AR; (ii) the A_2BAR, and not the A₁AR or A₃AR, is principally responsible for adenosine-mediated degranulation of canine BR mastocytoma cells; and (iii) the BR cell A_2BAR couples to both Ca²⁺ mobilization and cAMP accumulation. Although A_2B receptors play a major role in the regulation of BR mast cell degranulation, multiple AR subtypes and G proteins may influence mast cell functions.