High affinity 5-HT binding sites (5-HT1) were labeled in vitro on mounted rat brain slices using [3H]5-HT as a radioligand. In the first stage of experimentation, the bound radioactivity was measured on slices by liquid scintillation count in order to define the biochemical characteristics of the binding. Saturation curves were drawn, as well as association and elution curves for a 2 nM radioligand concentration. The mean affinity constant of the specific binding (Kd) was found to be 2.9 nM. In the second stage, the experimental parameters giving optimum binding were applied to the frozen slices prepared exactly as for the biochemical approach in order to investigate the effects of degeneration of retinal axon terminals on the distribution of 5-HT1 sites in the visual upper layers of the superior colliculus. The optical densities directly measured from tritium-sensitive film clearly indicate that the ablation of one eye causes a progressive reduction in the binding in the contralateral, largely deafferented, stratum griseum superficiale (SGS); with a 24-day survival period, the reduction was about 35-40%. In the homologous region of the ipsilateral colliculus, the binding decreased by about 10-15%. It is concluded that at least two populations of 5-HT1 binding sites coexist in the visual collicular layers, one of which is probably located on the axon terminals of retinal afferents. The present results confirm a previous hypothesis based on iontophoretic data, according to which this monoamine is involved in retino-collicular transmission. As far as the retinofugal terminal binding sites are concerned, 5-HT seems to exert a presynaptic control on visual inputs.