Neurons containing tuberoinfundibular peptide of 39 residues (TIP39) constitute a rostro-caudally elongated group of cells in the posterior thalamus. These neurons are located in the rostral part of the subparafascicular nucleus and in the subparafascicular area, caudally. Projections of the caudally located TIP39 neurons have been previously identified by their disappearance following lesions. We have now mapped the projections of the rat rostral subparafascicular neurons using injections of the anterograde tracer biotinylated dextran amine and the retrograde tracer cholera toxin B subunit, and confirmed the projections from more caudal areas previously inferred from lesion studies. Neurons from both the rostral subparafascicular nucleus and the subparafascicular area project to the medial prefrontal, insular, ecto- and perirhinal cortex, nucleus of the diagonal band, septum, central and basomedial amygdaloid nuclei, fundus striati, basal forebrain, midline and intralaminar thalamic nuclei, hypothalamus, subthalamus and the periaqueductal gray. The subparafascicular area projects more densely to the amygdala and the hypothalamus. In contrast, only the rostral part of the subparafascicular nucleus projects significantly to the superficial layers of prefrontal, insular, ectorhinal and somatosensory cortical areas. Double labeling showed that anterogradely labeled fibers from the rostral part of the subparafascicular nucleus contain TIP39 in many forebrain areas, but do not in hypothalamic areas. Injections of the retrograde tracer cholera toxin B subunit into the lateral septum and the fundus striati confirmed that they were indeed target regions of both the rostral subparafascicular nucleus and the subparafascicular area. In contrast, TIP39 neurons did not project to the anterior hypothalamic nucleus. Our data provide an anatomical basis for the potential involvement of rostral subparafascicular neurons in limbic and autonomic regulation, with TIP39 cells being major subparafascicular output neurons projecting to forebrain regions.