The transport rates of radiolabeled ascorbic acid (AA), 3-O-methyl-D-glucose (mD-glu) and L-glucose (L-glu) from blood into the aqueous humor, corneal endothelium, and stroma compartments were studied in male Sprague-Dawley rats and English short-haired guinea pigs. In vivo pulse chase kinetic studies supplied transport entry rate constants, Ki(min−1), and used L-glu as a passive internal control. Results in the rat indicate that AA enters the aqueous humor at rates similar to L-glu and likely via simple passive diffusion. Rate constants for entry into endothelium for both L-glu and mD-glu were high, indicating a quick equilibrium with the aqueous humor. Endothelium AA levels continued to increase to levels higher than in the aqueous humor. Although all three test molecules quickly entered the endothelium, L-glu and mD-glu levels were soon found to decrease, whereas levels of AA or its metabolites remained high. AA entered the stroma of the rat cornea slower than either L-glu or mD-glu, suggesting that no special transport mechanism for AA is present. mD-glu moves into the stroma quickly, but high levels are not maintained. The guinea pig maintains AA levels in the cornea about 10 times higher than that of the rat by first accumulating AA in the aqueous humor and again by actively accumulating AA in the endothelium. Data at 24 h postbolus suggests that both the rat and guinea pig maintain high corneal AA levels for extended periods. Thus, whereas both glucose analogues enter and leave the cornea via the aqueous humor relatively quickly, A A appears to be actively taken up by the endothelium and thus maintained high in both species. Metabolic roles for ascorbate in the eye could conceivably be similar in both nocturnal rats and diurnal guinea pigs, even though the means of supply differ. Because the guinea pig does not produce AA, it conserves and stores a supply in the aqueous humor via active transport. The rat instead could rely on a steady supply of AA to meet intraocular needs