A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 184.108.40.206) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H+ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88-95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of KI for edrophonium, decamethonium and Al3+.