Incorporation of L-[3H]leucine into the arcuate and supraoptic nuclei of rat and mice hypothalamus was studied by EM autoradiography at 30 min after intracerebral and intravenous application, respectively. The analysis applied provides information about (1) the distribution and (2) the concentration of radioactivity in the various tissue compartments, in particular nerve and glial cells. In the supraoptic nucleus, the proportion of radioactivity present in neuronal perikarya markedly exceeds that in the neuropil. Similar amounts of radioactivity in neuronal perikarya and neuropil were found for the arcuate nucleus. In both hypothalamic nuclei, only a small percentage of radioactivity is localized in glial cell bodies. However, the concentration of radioactivity in glial cells is nearly as high as in nerve cells and is lowest in the neuropil. Furthermore analysis of the subcompartments of the neuropil reveals that a considerable proportion of its radioactivity can be attributed to astrocytic processes. Conditions are discussed under which grain densities can be taken as measurements of relative synthesis rates of proteins. The results which are at variance with earlier autoradiographic studies are largely consistent with those of more recent biochemical studies on brain tissue separated into neuronal and glial fractions.