The morphology and projections of neurons in the paraventricular organ (PVO) were studied by means of silver impregnation after intraocular application of cobaltous lysine in the lungfish Protopterus dolloi. Cobalt-labeled neurons were found exclusively in the PVO in the dorsal and infundibular hypothalamus. These bipolar neurons possess one CSF-contacting process that protrudes into the ventricular lumen with a club-shape ending and a thick, ramifying process directed into the hypothalamic neuropil; the ependymofugal processes form intra- and extrahypothalamic projections. Impregnated fibers from paraventricular neurons cross in infundibular and hypothalamic commissures, the commissure of the posterior tuberculum, the postoptic, the habenular, and the anterior commissures. Projections to the infundibulum and the median eminence are relatively sparse; no fibers are labeled in the pituitary gland. Ascending projections to the forebrain are extensive. Major targets include the dorsal hypothalamus, the periventricular preoptic nuclei, the habenula, the subhabenular region, the anterodorsal thalamus, and the medial telencephalic hemisphere (septum). Most ascending fibers follow the medial forebrain bundle; others course in the fasciculus retroflexus and terminate in rostral parts of the ipsilateral habenula. Descending fibers run caudally along the ventral floor of the brainstem. They terminate in the neuropil of the mesencephalic tegmentum, ventral tectum, isthmic region, ventral portions of the reticular formation throughout the rhombencephalon, and extend into the spinal cord. Intraocular application of cobaltous lysine results in selective impregnation of neurons in the PVO and their ascending and descending projections, presumably via uptake of tracer from vascular circulation. These projections do not represent retinofugal or retinopetal projections. We provide conclusive evidence for the existence of a PVO in Protopterus. On the basis of PVO location and acetylcholinesterase histochemistry, we propose subdivisions of the infundibular hypothalamus corresponding to those in amphibians. Ascending PVO projections appear to be particularly well developed in lungfish compared with other species and may be related to specialized endocrine mechanisms in this group of vertebrates.