5-Methoxytryptamine is a potent agonist of presynaptic 5-hydroxytryptamine autoreceptors modulating serotonin release in the central nervous system. This methoxyindole can be synthesized in the pineal gland, but its presence in vivo is still controversial, probably because of rapid catabolism by monoamine oxidase. An improved high-pressure liquid chromatography method, with coulometric detection, has been developed for the simultaneous measurement of melatonin, 5-methoxytryptamine, 5-methoxytryptophol and 5-methoxyindolacetic acid. We have demonstrated a day-night rhythmicity in the amount of 5-methoxytryptamine in the pineal gland of golden hamsters (Mesocricetus auratus) maintained under a long photoperiod (14 h light: 10 h darkness) and pretreated with the monoamine oxidase inhibitor pargyline. Levels of 5-methoxytryptamine were highest at 16.30 h and lowest at 00.30 h. The rhythm for 5-methoxytryptamine appears to be the same as for serotonin (opposite in phase to that of melatonin). The identification of 5-methoxytryptamine has been confirmed by analysis with gas chromatography-mass spectrometry.