Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study we examined the domain specificity of recruitment and the role of soluble N-ethyl maleimide attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line, T84, was used as it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation and electron microscopic studies showed that the A2b receptor is intracellular at rest and apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing VAMP-2. Further, in cells stimulated with apical or basolateral adenosine we demonstrate a complex consisting of VAMP-2, SNAP-23 and A2b receptor that is co-immunoprecipitated in cells stimulated with adenosine within 5 minutes and is no longer detected in 15 minutes. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98% and 90% respectively. cAMP synthesis induced by foskolin was not affected suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that i) A2b receptor is intracellular at rest ii) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane iii) the SNARE proteins, VAMP- 2 and SNAP-23 participate in the recruitment of the A2b receptor and iv) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.