The location and chemical identity of neurons interconnecting the lateral geniculate complex and the hypothalamus were analyzed in order to provide further information on the anatomical substrates for the entrainment of circadian rhythms. A particular objective of the study was to characterize the neurons projecting between the intergeniculate leaflet (IGL) of the lateral geniculate complex and the suprachiasmatic nucleus (SCN) and related anterior hypothalamic areas. The connectivity experiments employed five combinations of fluorescent tracer injection and were combined with immunohistochemical localization of either neuropeptide Y (NPY), met-enkephalin (mENK) or the vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI) group. IGL efferents. Injection of tracer into the SCN results in retrograde labeling of NPY-immunoreactive neurons in the IGL as would be expected from prior work. These neurons and their terminals also contain the C-flanking peptide of the NPY precursor molecule (CPON). In addition, there are two additional groups of neurons in the IGL that project either to the SCN or the contralateral IGL but do not exhibit NPY immunoreactivity. These include a substantial population of cells that project to the SCN and an even larger group of neurons which project to the contralateral IGL and contain mENK immunoreactivity. Hypothalamic efferents. Injection of tracer into the IGL results in retrograde labeling of scattered neurons throughout the SCN and immediately adjacent anterior hypothalamus ipsilaterally and also in labeling of a small number of neurons in the same areas on the contralateral side of the brain. In rare instances, individual SCN neurons appear to project to both IGLs. However, the retrochiasmatic area (RCA) contains the largest number of retrogradely labeled neurons following tracer injections into the IGL. These neurons are concentrated along the midsagittal plane and in the lateral RCA ipsilateral to the injected IGL. None of the labeled neurons in the SCN or adjacent anterior hypothalamus exhibit VIP or PHI immunoreactivity. These observations indicate that the anatomical relations between the geniculate complex and the anterior hypothalamus are more complex than previously shown. First, the geniculohypothalamic tract arises from two distinct groups of IGL neurons: one contains NPY/CPON immunoreactivity; the chemical content of the other is not characterized at the present time. Second, the commissural projection between the two IGLs is formed by a third group of neurons, and these cells contain mENK immunoreactivity. Finally, reciprocal projections from the hypothalamus to the IGL arise from neurons in the retrochiasmatic area, SCN, and adjacent anterior hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)