Prostaglandin G and H synthases, or cyclooxygenases (COXs), catalyze the formation of prostaglandins (PGs). Whereas COX-1 is diffusely expressed in lymphoid cells in embryonic day 15.5 thymus, COX-2 expression is sparse, apparently limited to stromal cells. By contrast, COX-2 is predominant in a subset of medullary stromal cells in three- to five-week-old mice. The isozymes also differ in their contributions to lymphocyte development. Thus, experiments with selective COX-1 inhibitors in thymic lobes from normal and recombinase-activating gene-1 knockout mice support a role for this isoform in the transition from CD4(-)CD8(-) double-negative (DN) to CD4(+)CD8(+) double-positive (DP). Concordant data were obtained in COX-1 knockouts. Pharmacological inhibition and genetic deletion of COX-2, by contrast, support its role during early thymocyte proliferation and differentiation and, later, during maturation of the CD4 helper T-cell lineage. PGE2, but not other PGs, can rescue the effects of inhibition of either isoform, although it acts through distinct EP receptor subtypes. COX-dependent PG generation may represent a mechanism of thymic stromal support for T-cell development.