In this study, the P2X(5) receptor was found to be distributed widely in the rat hypothalamus using single and double labeling immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. The regions of the hypothalamus with the highest expression of P2X(5) receptors in neurons are the paraventricular and supraoptic nuclei. The intensity of P2X(5) immunofluorescence in neurons of the ventromedial nucleus was low. 70-90% of the neurons in the paraventricular nucleus and 46-58% of neurons in the supraoptic and accessory neurosecretory nuclei show colocalization of P2X(5) receptors and arginine vasopressin (AVP). None of the neurons expressing P2X(5) receptors shows colocalization with AVP in the suprachiasmatic and ventromedial nuclei. 87-90% of the neurons in the lateral and ventral paraventricular nucleus and 42-56% of the neurons in the accessory neurosecretory, supraoptic and ventromedial nuclei show colocalization of P2X(5) receptors with neuronal nitric oxide synthase (nNOS). None of the neurons expressing P2X(5) receptors in the suprachiasmatic nucleus shows colocalization with nNOS. These findings provide a morphological basis for possible functional interactions between the purinergic and nitrergic or vasopressinergic neurotransmitter systems.