Comparison of in vivo radiolabeled corneal proteoglycans from vitamin A deficient, pair-fed control and normal rabbits by chromatographic and enzymatic methods, reveals subtle but reproducible changes in the proteoglycans present in the vitamin A deficient corneas relative to those of pair-fed and normal control corneas. Although the total amounts of [3H]leucine and [35S]sulfate incorporated in vivo into the proteoglycans per cornea are similar in the three types of cornea, the proteoglycan affinity for DEAE-Sepharose is different, with more of the vitamin A deficient proteoglycans requiring a higher NaCl concentration for elution. Analysis of in vivo labeled rabbit corneal proteoglycans reveals that in vitamin A deficient animals, relative to pair-fed controls, there is (1) an increase in the proteoglycans digested by chondroitinase AC indicative of decreased epimerization of glucuronic acid to iduronic acid; (2) an increase in the amount of keratan sulfate proteoglycan with high affinity for an anion exchange resin, consistent with a greater negative charge; (3) differences in proteoglycan affinities for octyl-Sepharose, reflecting differences in hydrophobicity; (4) increased susceptibility to proteolysis by trypsin; (5) no significant difference in sulfation.