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A sensitive and specific enzymatic-isotopic micromethod for the determination of serotonin in animal tissues is described. The assay can measure as little as 50 pg of serotonin. Organ extracts are incubated with acetyl coenzyme A and rat liver N-acetyltransferase (ES 2.3.1.5) to acetylate seratonin and then incubated with ³H-methyl-S-adenosyl-1-methionine and pineal hydroxyindole-O-methyltransferase (ES 2.1.1.4), an enzyme that specifically O-methybates N-acetylserotonin. Under the conditions of the assay, ³H-melationin is the only radioactive product formed in detectable amounts from substrates extracted from tissue. Melatonin is produced in direct proportion to the serotonin added to the incubation mixture. With this assay, the endogenous serotonin content of small rat brain regions and nuclei can be measured.