It is not known if, in polarized cells, desensitization events can be influenced by the domain on which the receptor resides. Desensitization was induced by 5′-( N-ethylcarboxamido)adenosine (NECA) and was quantitated by measurement of short-circuit current ( I _sc) in response to adenosine. NECA added to either the apical or basolateral compartments rapidly desensitized receptors on these respective domains. Although apical NECA had no effect on the basolateral receptor stimulation, basolateral NECA induced a complete desensitization of the apical receptor. We hypothesized that desensitization of apical receptor by basolateral desensitization could relate to a trafficking step in which A2b receptor is first targeted basolaterally upon synthesis and transported to the apical surface via vesicular transport/microtubules. Because desensitization is associated with downregulation of receptors, apical adenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited I _scinduced by apical and not basolateral adenosine. In conclusion: 1) a single receptor subtype, here modeled by the A2b receptor, differentially desensitizes based on the membrane domain on which it is expressed, 2) agonist exposure on one domain can result in desensitization of receptors on the opposite domain, 3) cross-domain desensitization can display strict polarity, and 4) receptor trafficking may play a role in the cross-desensitization process.