The effects of electrophoretically applied prostaglandin D2 (PGD2) on neuronal activity in the rat lateral preoptic area (LPOA) and posterior hypothalamic area (PHA) were examined. In the LPOA, 20% of the tested neurons were excited, 26% inhibited, and 6% showed bidirectional response. The direct effects often showed desensitization after repeated applications. Neurons excited by PGD2 were significantly sensitive (excitation) to acetylcholine (ACh). The ACh excitatory effect was sometimes (38%) attenuated, blocked, or reversed by concurrent PGD2 application. Excitatory or inhibitory effect of noradrenaline (NA) was not related to the effects of PGD2; however, modulation of the NA responses by PGD2 was common (58%). Inhibition, the predominant NA response, was changed to no effect or to excitation during simultaneous PGD2 application. Changes of the NA responses from inhibition to excitation, or from excitation to inhibition-excitation sequences were observed after PGD2 infusion into the third cerebral ventricle at low concentrations. In 43% of the cells, neurotransmission in the LPOA following ventral noradrenergic bundle stimulation was modified by PGD2 application. PGD2 application tended to reduce the duration of inhibition and to extend that of excitation. The direct effects of PGD2 in the PHA were similar to those in the LPOA. Desensitization was also observed in the PHA, but no interrelations were observed among the effects of PGD2, ACh, and NA. Modulation of ACh and NA responses by PGD2 was rarely seen in the PHA. Possible contributions of PGD2 to sleep and thermoregulation are discussed.