A continuous spectrophotometric assay for phospholipase A2 (PLA2) was developed using novel carbonothioate phospholipids. These phospholipid analogues contain a carbonothioate bond in the place of the sn-2 ester of the natural substrates of phospholipase A2 and were synthesized in a one-pot two-step reaction. Phospholipase A2 from cobra venom (Naja naja atra) hydrolyzes carbonothioate phospholipids and liberates a free thiol, alkylmercaptan, which is reacted with 5,5'-dithiobis(2-nitrobenzoic acid) to yield a product that absorbs at 412 nm. The kinetic studies on PLA2 hydrolysis of carbonothioate phospholipids were carried out in pure phospholipid forms and in Triton X-100 mixed micelles. The hydrolysis of pure carbonothioate phospholipids exhibits an interfacial activation phenomenon. The hydrolysis of phospholipid in mixed Triton X-100 micelles follows classical Michaelis-Menten kinetics. In a mixed micellar system, the catalytic efficiency observed with this series of substrates is two orders of magnitude lower than that of the hydrolysis of the natural substrate dipalmitoyl phosphocholine. However, these substrates bind to the enzyme over 10 times tighter than does the natural substrate. Application of this carbonothioate assay to screen both reversible and irreversible enzyme inhibitors of phospholipase A2 is also demonstrated.