Extracellular degradation of adenosine by adenosine deaminase was studied in the rat duodenum using high performance liquid chromatography (HPLC) and pharmacological techniques. 2. Relaxant responses to adenosine (1-10 microM) were potentiated in a concentration-dependent manner by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin, both of which are inhibitors of adenosine deaminase. 3. Breakdown of adenosine, determined by HPLC, due to incubation with segments of rat duodenum was inhibited by both EHNA and deoxycoformycin. Cytosolic sources of adenosine deaminase were excluded. 4. Relaxant responses to adenosine were also potentiated by the adenosine transport inhibitor dilazep, which did not inhibit adenosine deaminase activity. 5. The extracellular adenosine deaminase activity (4 units/g tissue) was high compared with activity previously determined in other organs.