To study melatonin secretion by a gentle noninvasive method, a simple and quick RIA procedure to analyze melatonin in small urine volumes has been developed. Urinary extracts were prepurified by alkaline washes, and melatonin content was determined by RIA. The specificity of urinary melatonin determinations was confirmed by both thin layer chromatography and by gas chromatography-mass spectrometry. In the present study, we compared the amount of melatonin excreted in urine with plasma levels in 140 specimens from 13 adult volunteers. Comparisons showed a very good correlation between plasma levels at midnight and nocturnal excretion of urine, indicating the biological relevance of melatonin determination in urine. Our studies show further that urinary melatonin excretion displays the characteristic circadian rhythm usually observed in plasma. On the average, melatonin excretion is greatest between 2300--0300 h. The total 24-h excretion of melatonin varies considerably among different individuals.