Cyclooxygenase catalysis by prostaglandin H synthase-1 and -2 (PGHS-1 and -2) requires activation of the normally latent enzyme by peroxide-dependent generation of a free radical at Tyr-385 (PGHS-1 numbering) in the cyclooxygenase active site; the Tyr-385 radical has also been linked to self-inactivation processes that impose an ultimate limit on cyclooxygenase catalysis. Cyclooxygenase activation is more resistant to suppression by cytosolic glutathione peroxidase in PGHS-2 than in PGHS-1. This differential response to peroxide scavenging enzymes provides a basis for the differential catalytic regulation of the two PGHS isoforms observed in vivo. We sought to identify structural differences between the isoforms, which could account for the differential cyclooxygenase activation, and used site-directed mutagenesis of recombinant human PGHS-2 to focus on one heme-vicinity residue that diverges between the two isoforms, Thr-383, and an adjacent residue that is conserved between the isoforms, Asn-382. Substitutions of Thr-383 (histidine in most PGHS-1) with histidine or aspartate decreased cyclooxygenase activation efficiency by about 40%, with little effect on cyclooxygenase specific activity or self-inactivation. Substitutions of Asn-382 with alanine, aspartate, or leucine had little effect on the cyclooxygenase specific activity or activation efficiency but almost doubled the cyclooxygenase catalytic output before self-inactivation. Asn-382 and Thr-383 mutations did not appreciably alter the Km value for arachidonate, the cyclooxygenase product profile, or the Tyr-385 radical spectroscopic characteristics, confirming the structural integrity of the cyclooxygenase site. The side chain structures of Asn-382 and Thr-383 in PGHS-2 thus selectively influence two important aspects of cyclooxygenase catalytic regulation: activation by peroxide and self-inactivation.