The peroxidase associated with prostaglandin cyclooxygenase in ram seminal vesicle microsomes will utilize a wide variety of hydroperoxides and reducing substrates. One such reducing substrate, sulindac sulfide (cis-5-fluoro-2-methyl-1-[p-(methylthio)benzylidenyl]indene-3-acetic acid), inhibits the oxygenase, stimulates the peroxidase, and is oxidized to its analogous sulfoxide by the peroxidase. The peroxidase-catalyzed transfer of oxygen atoms from 15-hydroperoxyprostaglandin E2 (15-HPE2) to sulindac sulfide was examined using [18O]15-HPE2 which was prepared enzymatically and analyzed mass spectrometrically. The sulfoxide resulting from sulindac sulfide oxidation was also analyzed mass spectrometrically and found to possess an oxygen atom arising exclusively from the 15-HPE2. Since sulindac sulfide inhibits the oxygenase activity of this enzyme (ID50 approximately equal to 0.2 microM), it seemed possible that the oxygen atom was transferred while the sulfide was bound to this site. However, indomethacin, an inhibitor of the oxygenase with no effect on the peroxidase, did not alter the stoichiometry of sulindac sulfide oxidation, precluding this possibility. These findings are discussed in the context of identifying the nature of the actual oxidant and distinguishing between the oxidation mechanisms of various peroxidases and between sulindac sulfide and other reducing substrates for these enzymes.