Eighteen compounds related to thiamine were assayed for their inhibitory potency against electric eel acetylcholinesterase at pH 7.00 and 8.25 by Ellman's method. Data in the form of progress curves were fitted to the integrated form of the rate equation for linear, mixed-type Michaelis-Menten kinetics. The values of Ki thus obtained were compared in order to define the loci of inhibition on the thiamine molecule. It was found that the positively charged quaternary nitrogen atom is the primary locus of inhibition, while alkyl groups in the molecule play a secondary role through hydrophobic association with the enzyme. Protonation of the inhibitors was seen to be an important factor.