Prostanoids are involved in a wide range of biological processes including vascular function, wound healing, and inflammation. Prostaglandin endoperoxide synthases (PGHSs) generate the central precursor molecule for all prostanoids. New insights into the regulation of PGHS activity and isoform-specific functions in cells and in vivo have been gained over the past decade. Biophysical and mutagenesis studies have provided a detailed mechanistic and structural understanding of the regio- and stereochemical specificity of the reactions catalyzed by PGHS with arachidonic acid. Other lipids, including ω-3 fatty acids, oxidized fatty acids, and endocannabinoids, have been identified as alternative substrates for PGHS and, in many cases, are converted to novel eicosanoids that exhibit potent biological activities that are distinct from arachidonate-derived prostanoids.