The lateral preoptic and lateral hypothalamic regions contain the majority of the cell groups embedded in the fibre trajectories of the medial forebrain bundle on its course through the hypothalamus. Recent studies have extended considerably the parcellation of the lateral hypothalamic region, and therefore, the need to emphasize new insights into the anatomical organisation of projections from the neurons of the lateral hypothalamic region. In the present study we describe the anatomical organisation of efferent projections from the lateral preoptic and lateral hypothalamic regions to the hypothalamic paraventricular nucleus (PVN) on the basis of retrograde- and anterograde-tracing techniques. Iontophoretic injections of the retrograde tracer, cholera toxin subunit B, into the PVN revealed that most hypothalamic nuclei project to the PVN. Within the lateral hypothalamic region, retrogradely labelled cells were concentrated in the intermediate hypothalamic area, the lateral hypothalamic area, and the perifornical nucleus, whereas fewer retrogradely labelled cells were found in the lateral preoptic area. To determine the distribution of terminating fibres in subnuclei of the heterogeneous PVN, iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were delivered into distinct areas of the lateral hypothalamic region. Neurons of the intermediate hypothalamic area projected mainly to the PVN subnuclei, which contained parvicellular neuroendocrine cells. In contrast, neurons of the rostral and tuberal parts of the lateral hypothalamic area and the perifornical nucleus projected to the PVN subnuclei, which contained parvicellular neurons that send descending projections to preganglionic cell groups in the medulla and spinal cord. The perifornical nucleus was the only area within the lateral hypothalamic region that consistently innervated magnocellular perikarya of the PVN. Finally, all areas of the lateral hypothalamic region contributed substantially to fibres terminating in the perinuclear shell of the PVN. These results demonstrate that anatomically distinct areas of the lateral hypothalamic region have distinct projections to subnuclei of the PVN and further substantiate the view that the lateral hypothalamic region as well as the PVN constitute anatomically and functionally heterogeneous structures.