Nereistoxin blocks nicotinic cholinergic transmission, but the exact mechanism by which this dithiolane blocks nicotinic acetylcholine receptors is not clear. One possibility is that nereistoxin is reducedin vivoand the resulting product, dihydronereistoxin, reduces a disulfide bond in the region of the receptor where acetylcholine binds. Other possibilities include that nereistoxin oxalate as supplied is not pure and contains dihydronereistoxin, or that nereistoxin acts as a simple competitive antagonist. We report here a method for simultaneous detection of both nereistoxin and dihydronereistoxin using π reversed-phase, ion-pair high-performance liquid chromatography with electrochemical detection. The standard curve for nereistoxin is linear to 400 pmol, and the detection limit is 10 pmol. The working electrode response is stable for up to 60 injections. Reduction of nereistoxin to dihydronereistoxin by NaBH4was 92% complete under the conditions reported here, but reoxidation occurs slowly upon standing at room temperature. No dihydronereistoxin was detected in commercial samples of nereistoxin oxalate.