The importance of cysteine residues in the cyclooxygenase activity of prostaglandin endoperoxide synthase (PGHS) was investigated using cysteine-specific reagents and site-directed mutagenesis. N-(7-Dimethyl-amino-4-methyl-3-coumarinyl)maleimide (DACM), a hydrophobic maleimide, inactivated both cyclooxygenase and peroxidase activities of apoPGHS in a time-dependent manner but did not affect holoPGHS. Heme titration experiments indicated that modification of apoPGHS with DACM prevented heme binding. Peptide mapping revealed that DACM modified Cys313, Cys512, and Cys540. N-Ethylmaleimide inactivated cyclooxygenase and peroxidase activities of holoPGHS in a time-dependent manner but did not affect apoPGHS. Peptide mapping demonstrated that N-ethylmaleimide reacted primarily with Cys313 in holoPGHS and with Cys540 in apoPGHS. Each of the 3 cysteines was changed to serine by site-directed mutagenesis, and the mutant proteins were expressed in COS-1 cells. The C512S mutant converted arachidonic acid to products to the same extent as wild-type PGHS. In contrast, the C313S and C540S mutants converted arachidonic acid to products to the extent of 10% of wild-type PGHS. These results indicate that Cys313, Cys512, and Cys540 are not essential for cyclooxygenase activity but that alteration of Cys540 or Cys313 dramatically decreases enzyme activity. Both residues are well removed from the cyclooxygenase and peroxidase active sites so our findings reveal that subtle changes, such as substitution of a single oxygen for sulfur atom as far as 30 A from the heme prosthetic group, can significantly alter enzyme activity.