Adenosine potentiates the release of preformed mediators such as β-hexosaminidase (β-hex) from stimulated mast cells by a mechanism that is poorly understood. Mouse bone marrow-derived mast cells incubated for 2 hours with nanomolar concentrations of 4β-phorbol 12β-myristate 13αacetate (PMA) exhibited a marked suppression of the ability of adenosine to augment β-hex release induced by specific antigen or the calcium ionophore, A23187. Antigen-induced mediator release itself was also suppressed by PMA, whereas PMA and A23187 caused a synergistic enhancement of β-hex release. Overnight PMA treatment of mast cells produced a similar hyporesponsiveness to adenosine as well as a striking inhibition of the secretory response to antigen or A23187. The generation of leukotriene C4 was unaffected by overnight PMA. The ability of adenosine to augment mast cell cyclic AMP concentrations was only modestly inhibited by 2-hour or overnight PMA exposure. PMA induced an enhancement of antigen-stimulated intracellular free calcium levels as determined utilizing fura-2, but the ability of adenosine to potentiate this calcium response was completely abrogated. Either protein kinase C activation or a PMA effect on adenosine receptor expression or recycling in some way abolishes the ability of adenosine to augment mediator release. The inability of PMA to affect LTC4 generation underscores the dissociation between preformed and newly generated mediator releases. These data suggest that protein kinase C activity may be a key component of the mechanism of action of adenosine in mast cells.