Exposure of isolated retinas to 30 μM D-aspartate, which is a substrate for all high affinity glutamate transporters, for 30 min, resulted in the accumulation of such D-aspartate into Muller glial cells but not glutamatergic neurons as evinced by immunocytochemistry for D-aspartate. Further incubation of such loaded retinas in physiological media, in the absence of D-aspartate, resulted in the slow release of accumulated D- aspartate from the Muller cells and its accumulation into populations of photoreceptors and bipolar cells. This result indicates that after initial transport into Muller cells, reversal of direction of transport of D- aspartate, and thus by inference glutamate, by GLAST, readily occurs. D- aspartate released by Muller cells was strongly accumulated into cone photoreceptors which are known to express GLT-1, and into rod photoreceptors which we demonstrate here to express the retina specific glutamate transporter EAAT5 (excitatory amino transporter 5). Populations of glutamatergic bipolar cells, which express GLT-1 also exhibited avid uptake of D-aspartate. We conclude that the Muller cell glutamate transporter GLAST is responsible for most of the initial glutamate clearance in the retina after its release from neurones. However, some glutamate is also returned from Muller cells, to neurons expressing GLT-1 and EAAT5, albeit at a slow rate. These data suggest that the role of neuronal glutamate transporters in the retina may be to facilitate a slow process of recycling glutamate back from Muller cells to neurons after its initial clearance from perisynaptic regions by GLAST. (C) 2000 Elsevier Science Ltd.