The forebrain distribution of axons showing serotonin-like immunoreactivity was studied in the North American opossum. Serotonergic innervation of the hypothalamus was extensive, particularly within the ventromedial nucleus, the periventricular nucleus and the rostral supraoptic nucleus. Serotonergic axons were also present within the fields of Forel and zona incerta, but they tended to avoid parts of the subthalamic nucleus. In the thalamus serotonergic innervation was dense within the midline nuclei (e.g. the central, intermediate dorsal and rhomboid nuclei) and the ventral lateral geniculate nucleus, but relatively sparse in some of the nuclei more readily associated with specific functions (e.g. the ventrobasal nucleus). Serotonergic axons innervate most areas of the rostral and dorsal forebrain. Areas containing the heaviest innervation included the interstitial nucleus of the stria terminalis and the lateral septal nucleus. Serotonergic innervation of the neocortex varied markedly from region to region and within different layers of the same regions. The retrograde transport of True Blue combined with immunofluorescence for localization of serotonin revealed that serotonergic axons within the forebrain arise mainly within the dorsal raphe and superior central nuclei, but that some originate within the midbrain and pontine reticular formation and the locus coeruleus, pars alpha. Neurons of the raphe magnus and obscurus also innervate the forebrain, but few of them are serotonergic. The use of horseradish peroxidase as a retrograde marker provided evidence that raphe projections to the forebrain are topographically organized. Our results suggest that serotonergic projections to the forebrain, like those to the spinal cord, are connectionally heterogeneous.