The influence of prodrug structure on specificity of glycoside/glycosidase based colon-specific drug delivery was studied by preparing nine steroid glycosides, measuring their relative lipophilicities, and hydrolyzing them with bacterial glycosidases from rat intestines. The 21-yl beta-D-glucosides and galactosides of dexamethasone, prednisolone, hydrocortisone, and fludrocortisone and the 21-yl beta-D-cellobioside of prednisolone were prepared by a modified Koenigs-Knorr reaction. The deacetylated glycoside prodrugs, along with the P-nitrophenyl derivatives of beta-D-glucoside, galactoside, and cellobioside, were subjected to hydrolysis by the contents of the rat stomach, proximal small intestine (PSI), distal small intestine (DSI), and cecum. All the prodrugs were hydrolyzed slowly by PSI and stomach contents, more rapidly by contents of the DSI, and most rapidly by cecal contents. This is the basis of the site-specific drug delivery reported earlier (Friend, D. R.; Chang, G. W. J. Med. Chem. 1984, 27, 261). Furthermore, the prodrugs themselves had very different susceptibilities to hydrolysis. Hydrolysis rates catalyzed by DSI contents decreased in the following order: prednisolon-21-yl beta-D-galactoside (10) greater than prednisolon-21-yl beta-D-glucoside (2) greater than prednisolon-21-yl beta-D-cellobioside (13) greater than dexamethason-21-yl beta-D-galactoside (9) greater than dexamethason-21-yl beta-D-glucoside (1). Hydrolysis of cellobioside 13 was only half that of glucoside 2 and one-fourth that of galactoside 10. Hydrolysis of all the prodrugs in cecal contents was rapid, with the exceptions of hydrocortison-21-yl beta-D-glucoside (5) and fludrocortison-21-yl beta-D-glucoside (7), which were hydrolyzed more slowly than the other glucoside prodrugs. Eadie-Hofstee plots for hydrolysis of the glucoside compounds suggested that bacterial beta-D-glucosidase activity in the colon may be more heterogeneous in nature than beta-D-galactosidase activity. Relative lipophilicities of the prodrugs and free steroids were compared by measuring their octanol-buffer partition coefficients (P). The logarithm of the P of cellobioside 13 (-0.56) was considerably lower than that of the other prodrugs, which ranged from 0.11 to 0.84. Log P of the free steroids ranged from 1.54 to 1.73. These relative rates of hydrolysis and relative lipophilicities, along with previously reported animal experiments, enable one to estimate the site specificity of glycoside prodrugs prior to extensive animal studies.