Rat brain extract was found to catalyse the formation of 5-hydroxyindoleacetic acid from L-5-hydroxytryptophan in the presence of α-ketoglutarate, pyridoxal phosphate, NAD+ and iproniazide as a monoamine oxidase inhibitor. 5-Hydroxyindoleacetic acid was not formed in the absence of α-ketoglutarate. The omission of NAD+ from the incubation mixture decreased 5-hydroxyindole- acetic acid formation. These findings show that 5-hydroxyindoleacetic acid was produced from L-5-hydroxytryptophan via a transamination pathway which does not contain 5-hydroxytryptamine but 5-hydroxyindolepyruvic acid as an intermediate. The physiological significance of this pathway is discussed.