Adenosine or its synthetic analogues were topically applied to the intestinal jejunum while steady-state blood flow was calculated in submucosal arterioles using video microscopy. Blood flow increased (220 or 130% of control) with the serosal application of 10−6M N-ethyl carboxamido adenosine (NECA, A2-selective agonist) or 2-chloro adenosine (2CA, nonselective agonist) but not with cyclohexyl adenosine (CHA, A1-selective agonist). The nonselective competitive antagonist, 8-phenyl theophylline, attenuated the response evoked by NECA. The mucosal application of 10−6M CHA caused blood flow decreases (81% of control), but neither NECA nor 2CA evoked a response. These observations suggest a mucosal diffusion barrier, so the concentrations of the analogues were raised one hundredfold. Serosal 10−4M CHA or NECA caused blood flow increases, but the effects were negligible with mucosal application, suggesting that the mucosa was indeed impermeable to these compounds. The responses evoked by 10−4M 2CA were similar on the serosa or mucosa (200–220% of control), submaximal (maximum = 400% of control at 10−3M), and not antagonized by 8-phenyl theophylline or by the cellular uptake inhibitor, nitrobenzyl-6-thio guanosine. In context with earlier studies, >10−6M 2CA probably evokes vasodilation that is not entirely mediated by extracellular receptors. Serosal adenosine (10−4M) caused submaximal bood flow increases (200% of control) that were not potentiated by nitrobenzyl-6-thio guanosine or another transport inhibitor, dipyridamole. In contrast, mucosal adenosine (10−4M) had no effect on blood flow unless the transport inhibitors were present, and even then, the blood flow increase was lower than that evoked by serosal adenosine. Overall, these results suggest that 1) adenosine caused intestinal arteriolar vasodilation by intracellular and extracellular mechanisms, 2) the extracellular mechanism is mediated, in part, by A2-receptors, and 3) cellular uptake in the mucosa as well as a diffusion barrier probably restricts the passage of adenosine and its analogues from the lumen to the interstitium.