Experiments designed to ascertain the site or sites within the brain where prostaglandins act to evoke hyperthermia were carried out. At an ambient temperature of 20 degrees C, microinjections of PGE1 (50-100 ng) or saline vehicle were made in restrained rats. The 272 sites tested formed a matrix encompassing much of the subcortical tissue rostral to the medulla. The sensitivity of each site to PGE1 was evaluated on the basis of the dose required to elicit a criterion level of effect and on the intensity of the effect evoked by a fixed dose. Injections of PGE1 into a sensitive site typically produced a rapidly developing, short lasting, monophasic rise in body temperature. Saline injected at these sites produced no initial hyperthermia. Almost all of the active sites were located within the anterior hypothalamic/preoptic region (AH/PO). The sites of greatest reactivity were found in the ventral aspect of the tissue lying between the anterior commissure and the optic chiasm. These data suggest that the AH/PO region is an important and probably the only supramedullary site of PGE1 action in the rat brain.