The effect of chronic atropine treatment was studied on thyrotropin releasing hormone (TRH) content of several brain areas in Wistar rats. Atropine produced TRH increases in the septal area, preoptic area and the hypophysis; this was observed when rats were killed immediately after the last dose, while a decrease was observed only in the hypophysis 48 h after the last atropine dose. TRH concentration in cerebrospinal fluid rose significantly after atropine withdrawal with respect to controls. Treatment with eserine, an acetylcholinesterase inhibitor, produced the same effect. These results indicate cholinergic participation in central TRH regulation.