Using gas chromatography-negative ionization mass spectrometry, plasma melatonin levels in pinealectomized and sham-operated rats were assessed. The pinealectomized rats consistently demonstrated an absence of plasma melatonin while the intact animals showed detectable amounts. This suggests that although melatonin may be formed in tissues other than a pineal gland, the contribution to plasma is of pineal origin. Thus, plasma melatonin levels can be used as a marker of circadian melatonin secretion by the pineal gland and of its beta-adrenergic regulation.