We have investigated morphologic and histochemical characteristics of serotonin-containing epithelial cells in tracheas from adult rabbits, using the Falck-Hillarp freeze-dried formaldehyde vapor technique. An intracellular formaldehyde-induced fluorescent substance was identified as serotonin by microspectrofluorometric techniques. Fluorescence microscopy and subsequent histochemical staining of the same sections demonstrated that serotonin-containing cells were argentaffin-, argyrophil-, and ferric ferricyanide-positive. The serotonin-containing epithelial cells were more numerous in ventral than in dorsal aspects of trachea. The number of detectable fluorescent cells was reduced after reserpine administration but was not affected by injecting the amine precursor L-dihydroxyphenylalanine (L-DOPA). The emission peak of the fluorophore was not significantly shifted after L-DOPA injections. The cells may regulate tracheobronchial-pulmonary function by releasing serotonin or other as yet unidentified biologically active substances.