The formaldehyde-induced fluorescence (FIF) of pineal sections of the rat was measured quantitatively under instrumental conditions causing minimal photodecomposition. Fluorescence measurements on different sections of one pineal gland showed only little variation. Also the difference between animal variation was small, which enabled us to use groups of five to six animals for appropriate determinations. The similarity in emission spectra obtained from the pineal gland and a 5-hydroxytryptamine (5-HT)-containing protein model and the results of pharmacologic specificity tests indicated that pineal FIF is derived from 5-HT. Pineal 5-HT seemed to be stored in two compartments: the pinealocytes and the sympathetic nerve fibers. Reserpine, a drug depleting the neuronal 5-HT pool selectively, induced about 30% decrease in chemically detectable 5-HT content, indicating that 30% of total pineal 5-HT content was stored in the nerve fibers, and 70% was stored in the pinealocytes. FIF was not affected by reserpine, suggesting that FIF measurements were largely independent of the neuronal 5-HT store. The relationship between pineal FIF and chemically detectable 5-HT content was investigated in groups of rats treated with drugs increasing or decreasing pineal 5-HT content. From each group, five pineal glands were prepared for microfluorimetrical studies; the remaining pineal glands were used for chemical determinations of 5-HT and protein content. The linearity of this relationship demonstrated that concentration-dependent quenching did not occur in the fluorescence measured. Therefore it was concluded that FIF might be used as a quantitative parameter for 5-HT stored in the pinealocytes.