Summary—The subcellular distribution of clathrin has been examined in developing hypothalamic neurons cultured in a chemically defined medium up to synapse formation (12–13 daysin vitro) and exposed, or not, to a depolarizing concentration of KCl (60 mM for 3 min) followed, or not, by a return to control KCl concentration (3 mM KCl for 3 min). Previous studies have shown that such treatments induce in synaptic boutons a rapid vesicle depletion followed by massive restoration. Using an enzyme immunoassay, we have compared the relative proportion of assembled and unassembled pools of clathrin as a function of exposure to depolarizing or repolarizing concentrations of KCl. In parallel we have localized clathrin at the electron microscopic level using immunoperoxidase. Clathrin concentration in culture is lower (0.36vs0.75%) and the proportion of unassembled clathrin is much higher than in the adult brain (82vs14%). These proportions were not affected by depolarizing or repolarizing treatments. Morphologically clathrin was exclusively detected in two neuron compartments: perikarya and synaptic boutons. In perikarya clathrin was localized as a thick coat on plasma membrane coated pits and in the Golgi zone on coated buds and vesicles, presumably located in atranscompartment. In synaptic boutons clathrin immunoreaction was found as an irregular thin rim around synaptic vesicles, whatever the polarization state of the cells, but coated vesicles were extremely rare. Taken together these findings raise the problem of the functional meaning and localization of the large unassembled pool of clathrin in such neurons and question its role in vesicular traffic in synaptic bout