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5-Methoxytryptophol (ML) is found in the pineal gland and is known to have biological activity especially as an antigonadotrophic agent, but methods have been lacking for its measurement in the circulation. A gas chromatography–mass spectrometry assay using a trimethylsilyl derivative has been developed for the routine measurement of ML in plasma. The assay is of great specificity and has a sensitivity of 20 pmol/l. Studies on the levels of pineal indoles in the circulation, however, have been hampered by the possibility that extraneous compounds are being cross-measured. Thus the specificity of the routine assay has been further validated by comparing it with an alternative assay system where all the major parameters were changed, i.e. derivatizing reagent, internal standard and mass number. Results that were obtained using both assay systems were closely comparable.