The present study aimed to test the hypothesis that a decrease in preoptic cAMP mediates fever. To this end, body core temperature (T_c) of unanesthetized, freely moving rats was monitored by biotelemetry before and after pharmacological modulation of the cAMP pathway, and cAMP levels in the anteroventral third ventricular region (AV3V), where the preoptic region (POA) is located, were determined. We observed that intra-POA administration of the cAMP agonist dibutyryl-cAMP (Db-cAMP, 40 μg) reduced T_c. PGE₂ (the proximal mediator of fever, 200 ng) raised T_c with a concomitant decrease in AV3V cAMP levels from 22.7±1.8 to 17.0±1.0 fmol/μg protein. Moreover, PGE₂-induced fever was impaired by the phosphodiesterase inhibitor aminophylline. In order to verify the interaction between the cAMP- and cGMP-dependent pathways in the POA, we then co-injected Db-cAMP and 8-Br-cGMP into the POA. As a result, 8-Br-cGMP augmented the drop in T_c evoked by Db-cAMP. Lastly, we observed that intra-POA co-microinjection of the protein kinase A inhibitor (Rp-cAMPS, 1 μg) with the protein kinase G inhibitor (Rp-cGMPS, 1 μg), mimicking the effects of reduced production of cAMP and cGMP, respectively, produced a fever-like response. In summary, the present data support that a decrease in the levels of cAMP and cGMP in the POA is associated with the genesis of fever.