The present study was undertaken to assess whether cimetidine alters the levels of endogenous prostaglandin (PG) E2 and 6-keto-PGF1 alpha in the rat stomach. When cimetidine was mixed in vitro with a suspension of dextran-coated charcoal, to which [3H]-PGE2 had been adsorbed, levels of free [3H]-PGE2 were increased by cimetidine at concentrations above 1.0 mM, because of preferable adsorption of this blocker to the charcoal. Dextran-coated charcoal is used for the separation of antibody-bound [3H]-PGs from the free labelled compounds in the radioimmunoassay of PGs. Addition of cimetidine to standard solutions of PGE2 shifted the PGE2 calibration curve upward. Thus, when PGs in cimetidine-containing samples were quantitated by reference to the normal calibration curve, the assessed levels of PG were lower than the actual levels. Removal of cimetidine from the assay samples was successfully achieved by use of SEP-PAK C18 columns. Rats were injected intraperitoneally with 100 mg/kg of cimetidine only once or with 20 mg/kg twice daily for 7 days. Using this cleaning method, we found that both the basal levels of PGE2 and 6-keto-PGF1 alpha and those levels increased by intragastric administration of 1.0 M NaCl solution did not differ between rats treated with cimetidine and those treated with vehicle. It can be, therefore, concluded that cimetidine does not affect the gastric PG production.