Geldanamycin (GA) is a potent anticancer antibiotic that inhibits Hsp90. Its potential clinical utility is hampered by its severe toxicity. To alleviate this problem, we synthesized a series of carbohydrate-geldanamycin conjugates for enzyme-specific activation to increase tumor selectivity. The conjugation was carried out at the C-17-position of GA. Their anticancer activity was tested in a number of cancer cell lines. The enzyme-specific activation of these conjugates was evaluated with beta-galactosidase and beta-glucosidase. Evidently, glycosylation of C-17-position converted GA to an inactive prodrug before enzyme cleavage. Glucose-GA, as positive control, showed anticancer activity with IC(50) of 70.2-380.9 nM in various cancer cells by beta-glucosidase activation inside of the tumor cells, which was confirmed by 3-fold inhibition using beta-glucosidase specific inhibitor [2,5-dihydroxymethy-3,4-dihydroxypyrrolidine (DMDP)]. Compared to glucose-GA, galactose- and lactose-GA conjugates exhibited much less activity with IC(50) greater than 8000-25 000 nM. However, when galactose- and lactose-GA were incubated with beta-galactosidase in the cells, their anticancer activity was enhanced by 3- to 40-fold. The results suggest that GA can be inactivated by glycosylation of C-17-position and reactivated for anticancer activity by beta-galactosidase. Therefore, galactose-GA can be exploited in antibody-directed enzyme prodrug therapy (ADEPT) with beta-galactosidase for enzyme-specific activation in tumors to increase tumor selectivity.