It has long been known that mammalian retinas metabolize glucose aerobically to lactic acid and carbon dioxide. The classical view holds that glucose is the primary substrate for energy metabolism in all retinal cells, and that photoreceptor cells have the highest rates of glycolysis and respiration. A different and more recent view is that the Müller cells are the principal, if not sole aerobic producers of lactate, which then serves as the primary fuel for the mitochondria in photoreceptor cells and other retinal neurons. In this paper, we have examined these two competing hypotheses in rat and guinea pig retinas by identifying the cellular sites of glucose uptake and phosphorylation via hexokinase by means of autoradiographic studies with 3H-2-deoxyglucose (3H-2DG). The rat retina serves as a vascular model and the guinea pig retina serves as an avascular model. Rat and guinea pig eyecups were incubated in oxygenated, bicarbonate-buffered media containing glucose in the presence of labeled and unlabeled 2DG. Biochemical measurements of lactate production and ATP content were made on rat retinas incubated with different concentrations of glucose and 2DG in order to establish the optimal condition for conducting the autoradiographic studies with 3H-2DG. The optimal substrate concentrations were 1mM glucose and 0.25 mM 2DG. Results showed that following incubation of dark-adapted rat eyecups for 1 hr in media containing 1mM glucose/0.25 mM 2DG and supplemented with 3H-2DG, the label was distributed throughout all the layers of the retina, from the ganglion cell layer to the retinal pigment epithelium, with denser label associated with the outer retina (photoreceptors) relative to the density of label in the inner retina, as evaluated by counts of silver grains in individual retinal layers. Exposure of rat eyecups to light did not alter the relative distribution of label, but did increase total grain counts by 70%. However, uptake of labeled 2DG, as measured by scintillation counting of radioactivity in trichloroacetic acid extracts, was not significantly different between light- and dark-adapted rat retinas. In guinea pig eyecups, labeled 2DG was distributed throughout all the retinal layers. Addition of 10mM lactate or pyruvate to the glucose/2DG media produced no measurable change in the density or distribution of label in the eyecups. Measurements of the activity of hexokinase in rat retinas revealed that this enzyme was present in both the mitochondrial and cytosolic fractions. The present results suggest that as long as the availability of ambient glucose is adequate, retinal neurons use glucose, rather than glial-derived lactate, as the major substrate for the production of high energy phosphates.