We previously demonstrated by light and electron microscopic autoradiography that Xenopus retinas incubated with [3H]mannose exhibit tunicamycin-insensitive radiolabeling of glycogen storage compartments, especially cone parabaloids. In the present study, we utilized biochemical methods to evaluate the identity of the material presumed to be [3H]glycogen in Xenopus retinas obtained from eyecups incubated under similar conditions. A crude glycogen-containing fraction was isolated, solubilized with 8 M urea, and purified by Sepharose CL-4B column chromatography. The retinal glycogen was hydrolyzed either chemically or with specific amylolytic enzymes, followed by Sephacryl S-200 column chromatography and HPLC of the hydrolysis products. Under the conditions employed, [3H]glycogen represented at least 10% of the total radiolabeled macromolecules. Hydrolysis of the [3H]glycogen released all of the radiolabel in the form of [3H]glucose, not [3H]mannose, which indicated that direct incorporation of [3H]mannose into glycogen had not occurred. [3H]Glucose was distributed throughout the glycogen molecule, not just in the outer tiers, which indicated that de novo glycogenesis had occurred. Furthermore, enzymatic isomerization of the glycogen-derived [3H]glucose with glucose isomerase yielded fructose with retention of tritium. This demonstrated that positions other than the C-2 carbon of glucose were radiolabeled. Analysis of the medium after several hours of incubation revealed the presence of 3H2O as the major radiolabeled compound. These results support the conclusion that the in vitro incorporation of [2-3H]mannose into retinal glycogen involves initial catabolism of the radiolabeled substrate and subsequent reincorporation of the label via gluconeogenesis into precursors utilized for de novo glycogenesis.