Solubilized rat liver mitochondria were subjected to polyacrylamide gel electrophoresis. The monoamine oxidase activity was localized directly on the gel with radioactive substrates (serotonin, benzylamine, and tyramine). Serotonin and tyramine monoamine oxidase activity separated in several bands which migrated to the anode and cathode whereas benzylamine activity was localized in one band. This band was demonstrated only when the electrophoresis was run from cathode to anode. Each one of tyramine and serotonin activities could be found devoid of the other two activities. Benzylamine activity could be separated from the serotonin activity though not from the tyramine activity. The detection of monoamine oxidase with the tetrazolium staining provided a localization of the enzyme activity which was different from that observed using radioactive substrates. These results, in accordance with those previously obtained by us with sucrose gradient electrophoresis of the same preparation, support the existence of different enzymes for the oxidative deamination of benzylamine and serotonin. On the other hand our results did not eliminate the possibility of overlapping substrate specificity of tyramine activity with those of serotonin and benzylamine activities.