Light microscopy autoradiographs of the rat hypothalamic median eminence were prepared after injection of high specific activity tritiated GABA and GABA structural analogs. Following intracardiac injection of labeled GABA with short (15 min) survival time, a dense accumulation of silver grains was observed over the external layer of the median eminence. The silver grains appeared much less numerous and randomly scattered over the internal layer. No conspicuously labeled cells could be detected in the median eminence. A similar pattern of labeling was observed after 10 min in vitro incubation of the median eminence with a low concentration (2.5 x 10(-7) M) of labeled GABA. Clusters of silver grains were also visible over the external layer following intraventricular injection of labeled GABA. In this latter case, however, other sites of labeling were revealed over the internal and ependymal layers. The dense labeling over the external layer with tritiated GABA was partially reduced by a simultaneous intracardiac injection of a 50-fold excess of non-radioactive cis-aminocyclohexane carboxylic acid--a reported preferential substrate for GABA neuronal uptake--but it was not displaced by a 2000-fold excess of non-radioactive beta-alanine--a reported specific substrate for GABA glial uptake. Intracardiac injection of triated beta-alanine led to a faint and even labeling over the entire median eminence with no preferential accumulation of silver grains over the various layers. Following intraventricular injection of labeled beta-alanine the tanycytes and their processes as well as numerous glial cells appeared heavily labeled. These results suggested that there exist cell elements in the external layer of the hypothalamic median eminence which are capable of accumulating exogenous GABA according to its neuronal uptake characteristics. Although the exact nature of these cells is not readily apparent at this stage of our investigations, these findings led us to speculate that there might be a subpopulation of GABAergic nerve endings in the vicinity of the primary plexus capillaries.