The connections of the subfornical organ (SFO) were investigated by using the HRP technique. Injections into the SFO labeled neurons in the medial septum, but not in lateral septum nor in the diagonal band nucleus. Labeled cells were observed in the median preoptic nucleus, below the ependyma of the third ventricle, in the dorsal preoptic region near the anterior commissure, and diffusely throughout the medial preoptic and anterior hypothalamic areas. Fibers were followed from the ventral stalk of the SFO. Precommissural fibers enter the median preoptic nucleus where many of them appear to terminate. Others continue on to the medial septum, the OVLT, the supraoptic nucleus, and suprachiasmatic nucleus. HRP injections into the median preoptic nucleus labeled many neurons in the SFO. Postcommissural fibers reach the hypothalamus by descending along the walls of the ventricle in the subependymal space, by traveling in the columns of the fornix and the medial corticohypothalamic tract, or by passing through the paraventricular nucleus of the thalamus. Some postcommissural fibers turn rostrally and enter the median preoptic nucleus while others join precommissural fibers bound for the supraoptic nucleus. More caudally directed fibers appear to innervate the paraventricular nucleus of the hypothalamus and the medial preoptic and anterior hypothalamic areas. HRP injections into the paraventricular nucleus of the hypothalamus labeled neurons in the SFO. These findings corroborate and extend previous work in describing neural connections between two brain regions that are important for fluid balance.