Competition binding studies, subcellular distribution, and in vitro autoradiography were employed to compare the binding in rat brain of [3H]tryptamine with two radioligands for monoamine oxidase (MAO), [3H]pargyline, and [3H]1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine ([3H]MPTP). The MAO inhibitors pargyline, clorgyline, and deprenyl all yielded biphasic competition curves versus [3H]tryptamine. At low concentrations, these drugs stimulated binding by protecting the radioligand from MAO oxidation; at considerably higher concentrations, they inhibited binding by direct competition at the [3H]tryptamine binding site. In subcellular distribution studies, [3H]tryptamine was localized preferentially to the synaptosomal fraction, whereas [3H]pargyline showed greater binding to the mitochondrial fraction. Equilibrium binding studies revealed that the potencies of a series of seven compounds at inhibiting [3H]tryptamine binding were completely different from their potencies at inhibiting [3H]MPTP binding. Finally, the autoradiographic distribution of [3H]tryptamine binding in rat brain was different from that of [3H]MPTP and [3H]pargyline. We conclude that the [3H]tryptamine binding site in rat brain is not equivalent to MAO.