Neuropeptide FF (NPFF), a morphine modulatory peptide, has been identified within discrete autonomic regions in the brainstem and hypothalamus. Triple fluorescence labelling was employed to identify collateral branching projections of NPFF neurons located within the nucleus tractus solitarius (NTS) and in the region of the hypothalamus between the dorsomedial and ventromedial hypothalamus. Injections of two retrograde tracers, rhodamine- and fluorescein-labelled latex microspheres into the pontine parabrachial nucleus (PBN) and the ventrolateral medulla resulted in labelling of NPFF neurons in the NTS that contained one (double-labelled) or both (triple-labelled) tracers. Within the NTS, most double- and triple-labelled NPFF neurons were localized at the level of the area postrema or just rostral to it and within the medial and dorsomedial subdivisions of the nucleus. Injections of tracers into the PBN and hypothalamic paraventricular nucleus revealed double- and triple-labelled NPFF neurons, a majority of which were located in a zone between the dorsomedial and ventromedial hypothalamus. These results indicate that NPFF neurons in the brainstem and hypothalamus may simultaneously transmit signals to their target nuclei in the brainstem and forebrain. This coordinated signalling may lead to synchronized responses of NPFF target sites and provide insights into the role of this peptide in cardiovascular and nociceptive responses.