"Suicide" inactivation accompanied catalysis by isolated prostaglandin I synthase. Inactivation occurred via a saturable, pseudo-first order process with an apparent binding constant K_i = 8 μM prostaglandin H₂ and an inactivation rate constant k_i = 0.06 s⁻¹. Enzymatic activity declined as an exponential function of substrate concentration and a linear function of product formation. A competitive inhibitor, 9,11-(methanoepoxy)-15(S)-hydroxy-prosta-5Z,13E-dienoic acid, protected the enzyme from inactivation. Prostaglandin H₁, an endoperoxide which is not a substrate, inactivated the enzyme less effectively than prostaglandin H₂. The differences between inactivation by prostaglandin H₂ and H₁, the protective effect of the competitive inhibitor, the quantitative similarity between K_m and K_i, and the dependence on catalysis all suggest that inactivation originates primarily from a transition-state intermediate, not from malondialdehyde formed by hydrolysis of prostaglandin endoperoxides. collectively, the data conform to criteria for a specific, mechanism-based process in which a common enzyme-substrate complex participates in two parallel reactions, one leading to turnover and the other to suicide inactivation. Inactivation accompanying catalysis by prostaglandin I synthase in intact endothelial cells was transient, consistent with the cellular capacity for de novo protein synthesis. Enzyme activity returned to the initial steady-state level within 15-20 min, suggesting that prostaglandin I synthase has a half-life ≤ 5 min.