In the brain preproglucagon expression is limited to a cluster of neurons in the caudal part of the nucleus of the solitary tract (NTS) as well as a smaller number of neurons that extend laterally from the NTS through the dorsal reticular area into the A1 area. These neurons process preproglucagon to glucagon-like peptide-1 (GLP-1), GLP-2, oxyntomodulin and glicentin. The neurons project mainly to the hypothalamus, where especially two nuclei involved in appetite regulation – the paraventricular (PVN) and dorsomedial (DMH) hypothalamic nuclei – are heavily endowed with GLP-immunoreactive nerve fibres. To gain further insight into this neurocircuitry, we injected the retrograde tracers cholera toxin, subunit B (ChB) and Fluorogold (FG) into the PVN and the DMH, respectively. Of thirty-five injected rats, six had successful injections that predominantly restricted within the boundaries of the PVN and DMH. Hindbrain sections from these rats were triple labelled for ChB, FG and GLP-2. A total of 24±1% of the PVN-projecting NTS-neurons contained GLP-2-ir whereas 67±4% of the DMH-projecting neurons were also stained for GLP-2, suggesting that the NTS-projections to the DMH arise mainly from preproglucagon neurons. Approximately 20% of backfilled cells in the NTS contained both retrograde tracers, therefore presumably representing neurons projecting to both the PVN and the DMH. The results of the present study demonstrate that the majority of the preproglucagon-expressing neurons in the NTS project in a target-specific manner to the hypothalamus. It is therefore possible that individual subgroups of GLP-containing neurons can mediate different physiological responses.