During the tapping of papaya latex for papain (EC 3.4.22.2), benzyl isothiocyanate is enzymatically produced from benzylglucosinolate, a major component of the latex fluid. Benzyl isothiocyanate inhibits papain hydrolysis of α N- benzoyl- l- arginine ethyl ester (Bz-Arg-OEt). Since the availability of the papain sulfhydryl group to 5,5′-dithiobis-2-nitrobenzoic acid (Nbs 2) is inversely related to the extensiveness of the isothiocyanate inhibition, and treatment by benzyl isothiocyanate reduces the affinity of activated papain to a mercurialSepharose column, it is proposed that the papain sulfhydryl reacts with the electrophilic functional group of this inhibitor. Fifteen isothiocyanates were selected and both the chemical reactivity and their effectiveness as papain inhibitors determined. Three factors are involved in the isothiocyanate-papain inhibition: (1) chemical reactivity of isothiocyanates, (2) presence or absence of an aromatic substitution, and (3) the spatial relationship of the aromatic moiety to the NCS group of isothiocyanates. These data further suggest the presence of an ‘aromatic site’ near the Cys-25 sulfhydryl group of activated papain. Based on the existing model of papain molecule, the imidazole group of His-159 coincides well with our proposed aromatic site. The prospects of using isothiocyanates as chemical probes for the study of chemical environment of active sites in other enzymes are briefly discussed.