The occurrence of cholinesterases (ChE) has been demonstrated in retinas of several mammalian species. Using BW284C51 and iso-OMPA as selective inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, it has been demonstrated that the rat retinal ChE is predominantly AChE. Therefore the kinetic nature of inhibition of the rat retinal AChE by BW284C51 was studied using acetyl-6-methylthiocholine (AMTCh) as a selective substrate of AChE. AChE activity of the rat retinal sonicates was assayed using AMTCh as the substrate in the presence of 5,5-dithiobis-2-nitrobenzoate and yellow 5-thio-2-nitrobenzoic anion was measured by the absorption at 412 millimicrons using a spectrophotometer. The substrate (AMTCh) was varied between 0.1 and 0.5 mM. The inhibitor concentrations used were 2.1 and 4.2 nM. Double-reciprocal plots between substrate concentrations and the velocities for the enzymatic hydrolysis of AMTCh in the presence and absence of inhibitor were constructed. This study gave the following results: BW284C51 was a potent inhibitor of the hydrolysis of AMTCh by rat retinal AChE (IC50, 5.2 nM). The nature of the inhibition was found to be competitive as the double reciprocal plots with and without the inhibitor crossed on the ordinate.