Adenosine has potent effects on both the cardiovascular and immune systems. Exposure of tissues to adenosine results in increased vascular permeability and extravasation of serum proteins. The mechanism by which adenosine brings about these physiological changes is poorly defined. Using mice deficient in the A₃ adenosine receptor (A₃AR), we show that increases in cutaneous vascular permeability observed after treatment with adenosine or its principal metabolite inosine are mediated through the A₃AR. Adenosine fails to increase vascular permeability in mast cell–deficient mice, suggesting that this tissue response to adenosine is mast cell–dependent. Furthermore, this response is independent of activation of the high-affinity IgE receptor (FcεR1) by antigen, as adenosine is equally effective in mediating these changes in FcεR1 β-chain–deficient mice. Together these results support a model in which adenosine and inosine induce changes in vascular permeability indirectly by activating mast cells, which in turn release vasoactive substances. The demonstration in vivo that adenosine, acting through a specific receptor, can provoke degranulation of this important tissue-based effector cell, independent of antigen activation of the high-affinity IgE receptor, supports an important role for this nucleoside in modifying the inflammatory response.