ATP and ADP activate functionally distinct G protein-coupled purinergic (P2Y) receptors. We determined the expression and function of adenine nucleotide-specific P2Y receptors on cord blood-derived human mast cells (hMCs). Human MCs expressed mRNA encoding the ADP-specific P2Y1, P2Y12, and P2Y13 receptors; the ATP/UTP-specific P2Y2 receptor; and the ATP-selective P2Y11 receptor. ADP (0.05-50 muM) induced calcium flux that was completely blocked by a P2Y1 receptor-selective antagonist and was not cross-desensitized by ATP. Low doses of ADP induced strong phosphorylation of ERK and p38 MAPKs; higher doses stimulated eicosanoid production and exocytosis. Although MAPK phosphorylation was blocked by a combination of P2Y1- and P2Y12-selective antagonists, neither interfered with secretion responses. Unexpectedly, both ADP and ATP inhibited the generation of TNF-alpha in response to the TLR2 ligand, peptidoglycan, and blocked the production of TNF-alpha, IL-8, and MIP-1beta in response to leukotriene D(4). These effects were mimicked by two ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), but not by adenosine. ADP, ATP, adenosine 5'-O-(3-thiotriphosphate), and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate each induced cAMP accumulation, stimulated the phosphorylation of CREB, and up-regulated the expression of inducible cAMP early repressor, a CREB-dependent inhibitor of cytokine transcription. Human MCs thus express several ADP-selective P2Y receptors and at least one G(s)-coupled ADP/ATP receptor. Nucleotides could therefore contribute to MC-dependent microvascular leakage in atherosclerosis, tissue injury, and innate immunity while simultaneously limiting the extent of subsequent inflammation by attenuating the generation of inducible cytokines by MCs.