In vivo formation of ascorbic acid 2-O-alpha-glucoside (AA-2G) in guinea pigs and rats given ascorbic acid (AA) orally in combination with maltose was examined. A metabolite of AA which has the same HPLC retention characteristics as authentic AA-2G was detected in the blood, urine and liver of guinea pigs 1-2 hr after their administration. The metabolite was isolated from the urine by chromatographic procedures and identified as AA-2G by its UV spectrum, non-reducibility, susceptibility to alpha-glucosidase hydrolysis, HPLC profile and elementary analysis. The same glucoside was also synthesized by rats and found in the urine, although it could not be determined qualitatively in the blood. AA-2G-forming activities of tissue homogenates from both animals were apparently correlated with their alpha-glucosidase activities and, moreover, both activities were completely inhibited by a specific neutral alpha-glucosidase inhibitor. Thus, we conclude that AA-2G is a possible metabolite produced by enzymatic alpha-glucosidation after a combined administration of AA and maltose to guinea pigs and rats.