Using a simple high performance liquid chromatographic method, we measured melatonin, N-acetyl-5-hydroxytryptamine (NAc-5HT), 5-hydroxytryptophol (5HTOL), 5-hydroxyindoleacetic acid (5HIAA), 5-hydroxytryptamine (5HT) and tryptophan in single rat pineal glands. 5-Methoxytryptophol and 5-methoxyindoleacetic acid were not detected at levels which have been reported previously. 5HTOL, 5HIAA and 5HT showed the opposite diurnal rhythm to NAc-5HT and melatonin. Light during the dark period lowered melatonin while increasing 5HTOL, 5HIAA and 5HT. As the peak 5HTOL levels were higher than the peak melatonin levels, the functional significance of 5HTOL deserves consideration. Tryptophan administration increased melatonin and 5HTOL as well as the other indoles.