The nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetahydropyridine (MPTP) is an excellent substrate and a weak inactivator of the flavoenzyme monoamine oxidase B (MAO-B). In an attempt to develop novel mechanism-based inactivators of MAO-B, we have synthesized analogs of MPTP bearing a variety of functional groups at either the N or the C(4) position and have examined their interactions with a purified MAO-B preparation isolated from beef liver. The substituents selected include allyl, propargyl, ethenyl, ethynyl, and cyclobutyl, that is, functionalities which were considered potential sources of enzyme generated electrophilic or radical intermediates that might alkylate and inactivate the enzyme. None of the C(4)-substituted compounds displayed significant enzyme inhibitor properties although some proved to be good substrates. In the N-substituted MPTP series only the 4-phenyl-1-propargyl analog was a good inhibitor. The time- and concentration-dependent inhibition of MAO-B displayed by this compound is consistent with a mechanism-based inactivation pathway and the catalytic mechanism currently held for monoamine oxidases. The results of these studies provide additional insights into the steric features of the active site of MAO-B and predict that the area in which the C(4) substituent of the tetrahydropyridine ring resides lacks a reactive nucleophilic group.