Cyclooxygenase (COX) catalysis by prostaglandin H synthase (PGHS) is a key control step for regulation of prostanoid biosynthesis. Both PGHS isoforms are integral membrane proteins and their substrate fatty acids readily partition into membranes, but the impact of phospholipids and lipid membranes on COX catalysis and the actions of COX inhibitors are not well understood. We have characterized the COX kinetics and ibuprofen inhibition of the purified PGHS isoforms in the presence of phosphatidylcholine (PC) with varying acyl chain structure and physical state. PC was found to directly inhibit COX activity, with non-competitive inhibition by PC monomers binding away from the COX active site and competitive inhibition by micellar/bilayer forms of PC due to sequestration of the arachidonate substrate. Competitive inhibition by native membranes was observed in a comparison of COX kinetics in sheep seminal vesicle microsomes before and after solubilization of PGHS-1. PC liposomes significantly increase the inhibitory potency of ibuprofen against both PGHS isoforms without changing the reversible character of ibuprofen action or requiring binding of PGHS to the liposomes. These results suggest a useful conceptual framework for analyzing the complex interactions among the PGHS proteins, substrates, inhibitors and phospholipid.