Comparing a direct radioimmunoassay for 6-sulfatoxymelatonin (aMT6s) with an established gas chromatographic/mass spectrometric method for 6-hydroxymelatonin, we found a good correlation r = 0.94 (P less than 0.001, n = 100). aMT6s was stable, both in urine and plasma samples, without preservative, for at least two years at -20 degrees C and for five days at room temperature. Urinary excretion of aMT6s showed considerable inter-individual differences; however, the aMT6s excretion of any one individual was consistent over a four-day period, as assessed by continuous collection from 18 normal volunteers. Total 24-h urinary excretion of aMT6s was significantly correlated with the area under the curve of the respective profiles for plasma melatonin (r = 0.75, P = 0.0002) and plasma aMT6s (r = 0.70, P = 0.0005) for 22 healthy volunteers. At 24:00 h and 03:00 h, sampling plasma at 30-s intervals provided no evidence for episodic secretion (in short pulses) of either melatonin or aMT6s.