In chronic gastric fistula (GF) rats, HCl and a hyperosmolal solution of polyethylene glycol (PEG) in the upper intestine inhibit pentagastrin-stimulated gastric acid secretion by different mechanisms, but their anatomic sites have not yet been established. In the present study GF rats were provided with Thiry-Vella loops of the duodenum and the bile and pancreatic ducts transplanted to the proximal jejunum, or with Thiry-Vella loops of the proximal jejunum. In the latter rats the duodenum was anastomosed as a blind loop to the jejunum to prevent any gastric juice from entering the duodenum. Duodenal loop perfusion with 0.20 M HCl inhibited the acid response to pentagastrin by 62%, but perfusion with 1200 mOsmol x kg-1 of PEG solution did not alter the response. In contrast, acidification of the proximal jejunal loop did not alter but hyperosmolality inhibited the response by 41%. The study shows that the mechanism for inhibition by intestinal acidification is confined to the duodenum and that for inhibition by hyperosmolality is located in the proximal jejunum--but whether only to the proximal part is unknown.