Liver 9,000 X g supernatants from guinea pigs, rabbits, and dogs could catalyze the oxidation of both sulindac sulfide and sulindac, whereas those from mice and rats could catalyze only the oxidation of sulindac sulfide. In guinea pigs, the sulindac sulfide oxidase activity was detected in the 9,000 X g supernatants of kidney and lung as well as liver, whereas the sulindac oxidase activity was detected only in the liver preparation. In addition, the former activity was located in both liver microsomal and cytosolic fractions, whereas the latter activity was located only in the microsomal fraction. Both sulindac sulfide and sulindac oxidase activities of guinea pig liver microsomes were inhibited by SKF 525-A, N-ethyl-maleimide, and potassium cyanide. However, carbon monoxide inhibited only the oxidation of sulindac. The microsomal sulindac oxidase activity was enhanced 4-fold by 3-methylcholanthrene treatment.