Microvascular degeneration is an important event in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity. Because oxidant stress abundantly generates thromboxane A₂ (TxA₂), we tested whether TxA₂ plays a role in retinal vasoobliteration of OIR and contributes to such vascular degeneration by direct endothelial cytotoxicity. Hyperoxia-induced retinal vasoobliteration in rat pups (80% O₂ exposure from postnatal days 5–14) was associated with increased TxB₂ generation and was significantly prevented by TxA₂ synthase inhibitor CGS-12970 (10 mg · kg⁻¹ · day⁻¹) or TxA₂-receptor antagonist CGS-22652 (10 mg · kg⁻¹ · day⁻¹). TxA₂ mimetics U-46619 (EC₅₀ 50 nM) and I-BOP (EC₅₀ 5 nM) caused a time- and concentration-dependent cell death of neuroretinovascular endothelial cells from rats as well as newborn pigs but not of smooth muscle and astroglial cells; other prostanoids did not cause cell death. The peroxidation product 8-iso-PGF₂, which is generated in OIR, stimulated TxA₂ formation by endothelial cells and triggered cell death; these effects were markedly diminished by CGS-12970. TxA₂-dependent neuroretinovascular endothelial cell death was mostly by necrosis and to a lesser extent by apoptosis. The data identify an important role for TxA₂ in vasoobliteration of OIR and unveil a so far unknown function for TxA₂ in directly triggering neuroretinal microvascular endothelial cell death. These effects of TxA₂ might participate in other ischemic neurovascular injuries.